Tuli Dey
MOLECULAR CHARACTERIZATION OF Entamoeba invadens
CHITINASES: AN ENCYSTATION SPECIFIC PROTEIN
of
Doctor of Philosophy
by
Tuli Dey
DEPARTMENT OF BIOTECHNOLOGY
JULY 2009
© 2009 Tuli Dey. All rights reserved.
Declaration
I certify that,
a. The work contained in this thesis is original and has been done by me under the
guidance of my supervisor, Dr. Sudip K. Ghosh.
b. The work has not been submitted to any other Institute for any degree or diploma.
c. I have followed the guidelines provided by the Institute in preparing the thesis.
d. I have conformed to the norms and guidelines given in the Ethical Code of Conduct
of the Institute.
e. Whenever I have used materials (data, theoretical analysis, figures, and text) from
other sources, I have given due credit to them by citing them in the text of the thesis
and giving their details in the references. Further, I have taken permission from the
copyright owners of the sources, whenever necessary.
(Tuli Dey)
ii
Acknowledgement
IIT Kharagpur
July 2009 (Tuli Dey)
iv
List of Symbols
˚C Degree Centigrade
4MU 4-methylumbelliferone
ABS Adult bovine serum
Amp Ampicilline
APS Ammonium per sulphate
AU Arbitrary Unit
BLAST Basic Local Alignment Search Tool
bp Base pair
BSA Bovine serum albumin
cDNA Complemenatary DNA
CFW Calcoflour white
Da Dalton
DAB Diaminobenzidine
DAPI 4′,6-Diamidino-2-phenylindole dihydrochloride
DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
DTT Dithiothreitol
E. coli Escherichia coli
EDTA Ethylenediaminetetraacetic acid
EGTA Ethylene glycol tetraacetic acid
Eh Entamoeba histolytica
Ei Entamoeba invadens
ELISA Enzyme-linked immunosorbent assay
EtBr Ethidium bromide
gm Gram
GalNac N-Acetylgalactosamine
GlcNac N-Acetylglucosamine
HPLC High-performance liquid chromatography
hr Hour
HRP Horseradish peroxide
Ig Immunoglobulin
IPTG Isopropyl β-D-1-thiogalactopyranoside
kana Kanamycin
KCl Potassium chloride
kDa KiloDalton
LB Luria broth
min Minute
ml Milliliter
mM Milimolar
mRNA Messenger ribonucleic acid
Mtz Metronidazole
N.D Not detected
NaCl Sodium chloride
ng Nanogram
nm Nanometer
nM Nanomolar
O.D Optical density
v
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline
PCR Polymerase Chain Reaction
pM Picomolar
PMSF Phenylmethylsulphonyl fluoride
rDNA Ribosomal DNA
RNA Ribonucleic acid
RNase Ribonuclease
rpm Rotation per minute
rRNA Ribosomal RNA
RT-PCR Reverse transcriptase Polymerase Chain Reaction
s Second
SDS Sodium dodecyl sulfate
SLS Sodium lauryl sulfate
Taq Thermus aquaticus
TEMED N,N,N',N'-Tetramethylethylenediamine
Tetra Tetracycline
TIGR The Institute for Genomic Research
TRITC Tetramethyl Rhodamine Isothiocyanate
UNICEF United Nations Children's Fund
UV Ultra violet
WHO World Health Organization
X-gal 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
YT Yeast Extract Tryptone
β-ME β-mercaptoethanol
µg Microgram
µl Microliter
µm Micrometer
µM Micromolar
vi
Abstract
Amoebiasis remains as a morbid disease throughout the tropical counties, due to the
unhygienic living conditions. Entamoeba histolytica (Eh), the causative agent is an
anaerobic protozoa completes its life cycle between the cyst and trophozoite stage.
Current post genomic knowledge of Eh was used to identify new drug targets within
different molecules, including encystation and excystation specific ones. Chitinase of
other parasites were used successfully as a drug target or for the antigen mediated
transmission blocking.
Detail molecular characterization of Entamoeba chitinases was necessary for
identifying exclusive and new drug target as several chitinases like proteins were
reported in human, with few conserved domains. Reptilian parasite Entamoeba
invadens (Ei) was used as a model organism for encystation study as Eh was not
amenable to encystation in axenic condition. Several encystation specific proteins
were categorized as potential drug targets, including chitinases, as its inhibitor was
able to restrain the encystation. Ei contains three chitinases, while Eh contains single
one.
In this study, three chitinases of Ei named as EiChit1, EiChit2 and EiChit3 were
characterized in silico, to identify the conserved aromatic residues, and substrate
binding mode. All Ei and Eh chitinases were cloned and expressed with N-terminal
His tag. Recombinant chitinases were found to have hydrolytic activity, and bound to
insoluble chitin, without any chitin binding domain present in EiChit2 and EiChit3.
Recombinant chitinases act as exochitinase; by cleaving dimers from the chitin
polymer. Several methylxanthine derivatives were found to inhibit the enzymes in
competitive or non-competitive way though in micro molar range.
Importance of ChBD of EiChit1 was determined by its replacement with lectin ChBD
and removal. Removal of ChBD was found to improve the enzymatic ability against
the soluble substrate. Fusion protein with lectin ChBD shows diminished activity.
Number and location of ChBD aromatic residues was found to influence the substrate
binding.
Initiation of encystation was instrumental in chitinase transcription, which was
profiled during encystation by RT-PCR. Localization of translated chitinases during
encystation was observed by fluorescence microscopy using polyclonal anti EiChit1
and anti EiChit2 antibody. EiChit2 antibody was able to identify Eh cysts due to the
sequence and structural similarity.
vii
CONTENTS
Title page i
Declaration ii
Acknowledgement iv
List of symbols v
Abstract vii
Contents viii
1.1 Introduction 3
1.2 Amoebiasis and Amoebic parasite 3
1.3 Entamoeba histolytica and its life cycle 6
1.4 Metronidazole and Metronidazole resistivity 7
1.5 Encystation and Excystation specific molecules as
probable drug candidate 9
1.6 Entamoeba invadens as a model for Entamoeba
histolytica encystation 9
1.7 Encystation of Entamoeba 10
1.8 Chitin and Chitosan 12
1.9 Chitinases 13
1.10 Chitin Binding domain of Chitinases and Chitin lectin 15
1.11 Objectives 16
2.1 Material 19
2.1.1 Entamoeba Strain 19
2.1.2 Bacterial Strain 19
2.1.3 Primer 20
2.1.4 Molecular Biology Material 21
2.1.5 Molecular weight marker 21
2.1.6 Immunodetection reagents 21
2.1.7 Chemicals 22
2.1.8 Instruments, Glass and Plastic wares 22
2.1.9 Recipes of medium, buffers and solutions 36
2.1.10 Kits 36
2.1.11 Vectors 36
2.2 Microbiological Methods 39
2.2.1 Growth of bacterial culture 39
2.2.2 Preparation of competent cell 39
2.2.3 Transformation 40
2.2.4 Selection of blue-white colony 40
2.2.5 Bacterial cell storage 40
viii
2.3 Molecular Biology Methods 41
2.3.1 Genomic DNA isolation 41
2.3.2 Total RNA isolation 41
2.3.3 Plasmid DNA isolation 42
2.3.4 cDNA synthesis 43
2.3.5 RT-PCR 44
2.3.6 PCR amplification from genomic DNA 44
2.3.7 Restriction enzyme digestion 44
2.3.8 Agarose gel electrophoresis for DNA and RNA 45
2.3.9 Purification of the gel eluted sample 45
2.3.10 Spectrophometric estimation of DNA and RNA 46
2.3.11 Ligation 47
2.3.12 Automated dye terminator cycling sequencing
of DNA 47
2.4 Protein method 48
2.4.1 Expression of recombinant His-tagged protein
under native conditions 48
2.4.2 SDS-PAGE 49
2.4.3 Coomassie blue R-250 staining and destaining 49
2.4.4 Silver staining 49
2.4.5 Purification of recombinant His-tagged protein
under native conditions 50
2.4.6 Regeneration of used column 50
2.4.7 Quantification of protein 51
2.4.8 Western blotting 51
2.4.9 Electroelution 52
2.4.10 ELISA 52
2.4.11 Enhanced chemiluminescence 53
2.4.12 Raising polyclonal antibody in rabbit 54
2.5 Enzymatic methods 55
2.5.1 Enzyme assay 55
2.5.2 Inhibitor assay 55
2.5.3 Substrate binding assay 56
2.5.4 Substrate cleavage pattern 56
2.6. Entamoeba cell culture methods 57
2.6.1 Entamoeba cell culture 57
2.6.2 Encystation of Entamoeba invadens 57
2.6.3 Excystation of Entamoeba invadens 57
2.6.4 Entamoeba total protein isolation 58
2.6.5 Fluorescence Microscopy 58
2.6.6 Cryopreservation of Entamoeba cell 59
2.6.7 Revival of cryopreserved cell 59
3.1 Abstract 63
3.2 Introduction 63
3.3 Results
3.3.1 Data mining and sequence identification 64
ix
3.3.2 Preliminary information regarding Entamoeba
chitinases 65
3.3.3 Phylogenetic analysis of Entamoeba chitinase 66
3.3.4 Primer designing 68
3.3.5 Homology modelling 70
3.3.6 Molecular docking 72
3.4 Discussion 73
3.5 Conclusions 75
4.1 Abstract 79
4.2 Introduction 79
4.3 Results
4.3.1 PCR amplification of Entamoeba chitinases
from genomic DNA 80
4.3.2 Cloning of Entamoeba chitinases in pGEMT
vector 81
4.3.3 Cloning of Entamoeba chitinases in pQE30
vector 82
4.3.4 Expression of Entamoeba chitinases in bacterial
system 83
4.3.5Purification of Entamoeba chitinases using
Nickel NTA beads 84
4.4 Discussion 86
4.5 Conclusions 88
5.1 Abstract 91
5.2 Introduction 91
5.3 Results
5.3.1 β (1-4) bond cleavage efficiency 93
5.3.2 Insoluble substrate binding efficiency 95
5.3.3 Insoluble substrate cleavage efficiency 97
5.3.4 Inhibitor assay using Methylxanthine compounds 98
5.4 Discussion 99
5.5 Conclusions 102
x
6.3.3 Construction of JacobChBD-EiChit1R-CAT and
JessieChBD-EiChit1R-CAT fragments 110
6.3.4 Cloning, Expression and purification of EiChit1CAT,
JacobChBD-EiChit1R-CAT and JessieChBD- EiChit1R-CAT
Fragments in pQE30 vector 112
6.3.5 β (1-4) bond cleavage efficiency of truncated and
fusion protein 113
6.3.6 Insoluble substrate cleavage efficiency of
JacobChBD-EiChit1R-CAT and
JessieChBD- EiChit1R-CAT 114
6.3.7 Substrate binding efficiency of
JacobChBD-EiChit1R-CAT and
JessieChBD- EiChit1R-CAT 116
6.4 Discussion 117
6.5 Conclusions 119
References 147-158
Appendices 159-164
xi
Chapter 1
Introduction
2
1.1 Introduction
Amoebic colitis and amoebic liver abscess were known to the ancients, as Hippocrates
recognised that “Dysenteries, when they set in with fever, alvine (intestinal)
discharges of a mixed character or with inflammation of the liver … are bad” (Stanley
Jr., 2003). More than 200 years elapsed after amoebas were identified as a cause of
dysentery, by the St Petersburg physician Fedor Aleksandrovich Losch in 1875
(Losch, 1978). In early literature the genus names Endamoeba and Entamoeba were
used to describe Entamoeba histolytica. As per the ruling of The International
Commission on Zoological Nomenclature from 1954 onwards, the E. coli was
designated to be the type species and the term Entamoeba was used in place of
3
Endamoeba to describe E. histolytica (Bruckner, 1992). Several members of amoebic
populations were found to dwell the human gastrointestinal system such as Entamoeba
dispar, Entamoeba hartmoni, Entamoeba coli, Endomalix nana, though only the E.
histolytica, an anaerobic parasite, found to be the sole responsible for this disease, and
use human as the only reservoir. This parasite evolved early and survived till today,
without any major physiological changes (Clark, 2000). The Entamoeba lineage has
no close relatives among free living protozoan. The conclusion from ribosomal DNA
and other genomic sequence analyses shows that the genus Entamoeba diverged from
other eukaryotes much later than other “more” eukaryotic cell (Fig1.1). This
speculation probably indicates the secondary loss of several eukaryotic structures in
response to the obligatory parasitic life cycle of this organism (Clark and Roger, 1995).
Fig. 1.1: The fragment comigration data derived from riboprint patterns were converted into
estimated genetic distances by the method of Nei and Li and the tree constructed by the Fitch-
Margoliash method (Clark and Diamond 1997)
4
infectious host for a short time only. Among the 48 million cases of probable infection
throughout the world, only 10% have found to show clinical symptoms with intestinal
and /or extra intestinal pathology (Walsh, 1986). Recently the division of E.
histolytica into pathogenic E. histolytica (invasive) and non-pathogenic E. dispar
(non-invasive) has been done depending on isoenzyme typing as well as gene
sequencing study.
These syndromes constitute the classic ambulatory dysentery and can easily be
distinguished from that of bacterial origin. Although E. histolytica can infect almost
every organ of the body, the most frequent form of extraintestinal amebiasis is the
amebic liver abscess. This condition, which results from the migration of trophozoites
from the colon to the liver through the portal circulation, is 10 times more common in
adults than in children and 3 times more frequent in males than in females (Sepulveda
et al., 1986). Dissemination of amoebic infection to extra-intestinal sites most
commonly involves the liver, lungs, pericardium, brain and skin through the
circulating system. Infections with E. histolytica may or may not be symptomic,
though in 20% of infected cases parasite invades beyond the gastrointestinal
periphery, which if left untreated, results in death.
5
1.3 Entamoeba histolytica and its life cycle
The life cycle of E. histolytica is simple and consists of two stages, the motile
invading stage known as trophozoites, and non-motile infecting stage termed as cyst.
Mature cysts ingested with contaminated food or water, pass through the acidic
environment of stomach and become active after entering the neutral or slightly
alkaline condition of small intestine. Four trophozoites (small metacystic trophozoites)
emerged from single cyst and developed into the normal trophozoites which become
established within the mucous layer of intestine.
6
karyosome (in stained sample). Mitochondria like structure or crypton or mitosome
and Golgi like structure are reported in some recent publications (Mai et al., 1999;
Ghosh et al., 2000). Developments of endoplasmic reticulum like structure are being
observed under electron microscopy (Teixeira et al., 2008).
Cyst- Immature cyst may contain a glycogen mass and a highly refractile
chromatoidal bodies with smooth rounded edges. In spherical mature cyst (metacyst)
the diameter varies from 10-20 µm (usually 12-15 µm), and the glycogen mass
disappears completely. Four well distinct nucleuses along with several chromatoidal
bodies are being found throughout the cytoplasm which is covered with a 20 nm thick
wall constituted with protein and polysaccharide mesh. The excystation occur in the
terminal ileum giving rise to four trophozoites from each cyst (Espinosa-Cantellano et
al., 2000).
Two classes of drugs are generally used depending on the localization of this parasite.
Paramomycin, Diloxanide fluroate, and Idoquinol act on the intestinal parasite and
classified as Luminal ameobicides, while metronidazole, chloroquine and
Dehydroemetine affect the tissue-invading amoebas, and named as tissue ameobicides.
Metronidazole (Mtz) (1-[2hydroxyethyl]-2-methyl-5-nitroimidazole) was first
introduced as trichomonicide in 1959 (Cosar et al., 1959). At present this drug is used
as a single treatment against several anaerobic bacterial and protozoan infections,
along with other derivatives of nitroimidazole [2-(2-methyl-5-nitro-1H-imidazol-1-yl)
ethanol] (Gillin, 1981). This prodrug is marketed under the trade name “Flagyl” and
gets activated selectively after the reduction of nitro group by several electron carriers
and reducing enzymes, within the anaerobic parasites (Fig. 1.3) (Upcroft et al., 2001,
Pal et al., 2009).
7
Fig. 1.3: Metronidazole structure. Black arrow indicates the position of reduction by which the
drug gets activated.
This unique feature of anaerobic activation made it a wonder drug against several
anaerobic bacteria and protozoa’s, as it remains inactive within highly aerobic
environment of eukaryotic cells. The reduced products are responsible for disrupting
the DNA structure and produce adduct structure with proteins, inhibiting the cell cycle
(Leitsch et al., 2007).
Extensive and indiscriminate use due to easy availability of Mtz and its derivatives is
lead it’s resistivity among different group of prokaryotes. Metronidazole resistance
has been reported in pathogenic bacteria like Helicobacter pylori (Goodwin et al.,
1998; Koivisto et al., 2004), Bacteroides spp. (Narikawa et al., 1991; Diniz et al.,
2004) and Clostridium difficile (Pelaez et al., 2008). Among protozoan parasites,
Trichomonas vaginalis, Trichomonas foetus (Johnson, 1993) and Giardia intestinalis
(Ellis et al., 1993) have shown resistance towards Mtz. Reports of cross-resistance of
Bacteroides spp against nitroimidazoles are well documented now, and becoming very
common (Haggoud et al., 2001). Current literature shows the growing trend of cross-
resistance against Mtz or 5-nitroimidazole family of drugs (Dunne et al., 2004), which
can lead to the failure of current drug therapy. Absence of proper species level
identification of Entamoeba in pathogenic or non-pathogenic carrier increase the risk
of unwanted drug exposure towards the non-pathogenic strains also. Sufficient
evidence is available to classify this drug as an animal carcinogen, as it shows against
rodent (in vivo and in vitro) (Trzos et al., 1978; A-Kareem et al., 1984) though its
genotoxicity has not been proved yet in human (Bendesky et al., 2002). In spite of
ambiguous data regarding human study, chronic and indiscriminated use of Mtz
renders its role as a possible carcinogen among individuals with Crohn’s disease
(Krause et al., 1985). This situation demands a new array of putative drug candidates
as it is frightening to imagine the consequences if large-scale resistance is to emerge.
8
1.5 Encystation and Excystation specific molecules as probable drug candidate
Cyst, the infectious form of E. histolytica, is a chitin walled dormant stage, which
propagate the infection. Excystation or the emergence of four rapidly diving
trophozoites from a cyst occurs in the alkaline environment of small intestine
(Eichinger, 1997). Encystation or the cyst formation occurs in the large intestine as a
result of unknown cues, which may range from low nutrients, high population or host
conditioning (Arroyo-Begovich et al., 1980). Removal of nutrient factors such as
serum and glucose from the culture medium results in the formation of multinucleated
cyst-like structures in E. histolytica, however complete encystation of E. histolytica
not yet been observed (Barron-Gonzalez et al., 2008).
This imposed trigger suggests that when the environment is not suitable for
trophozoite survival this protozoan enters the encystation procedure and probably this
process is irreversible. Thus disrupting the encystation process can lead to eventual
cell death due to nutrient depletion and high osmotic pressure. A chemotherapeutic
agent can be designed to block the encystation and can produces a fatal outcome for
the protozoan, which could at the very least serve to reduce transmission (Jarroll et al.,
2003).
Inhibition of cyst formation may curtail the infection of Entamoeba, and may help to
identify novel chemotherapeutic targets. With the completion of the E. histolytica
genome project (Loftus et al., 2005) chances of identification of potential therapeutic
or vaccine targets increased rapidly (Laughlin et al., 2005).
Till now no axenic synchronous encystation protocols have been reported for E.
histolytica, therefore, studies in this area have concentrated on a related reptilian
parasite Entamoeba invadens which have similar basic morphology and life cycle. E.
invadens exists within reptiles such as turtle and snakes as a commensal, but act as
pathogen among a subset of hosts and create a similar type of colon pathology like E.
histolytica (Donaldson et al., 1975).
9
Genome wide survey of E. invadens shows higher GC content than that of E.
histolytica. E. invadens gene sequences shows 62% sequence identity at the genome
level and 74% sequence identity at coding regions with E. histolytica genes, with a
high amount of laterally transferred genes of metabolic pathways as similar to E.
histolytica. Several highly repetitive sequences such as rRNAs, tRNAs, CXXC-rich
proteins, and Leu-rich repeat proteins are detected in E. histolytica genome and find to
be repetitive in E. invadens genome also. E. histolytica proteins involved in amoebic
virulence, drug resistance, cell cycle, vesicular trafficking, signal transduction,
en/excystation and cell growth have homologous components in E. invadens genome
confirming its candidature as a potential model of E. histolytica study (Wang et al.,
2003).
Encystation of several protozoan parasites was studied before in vitro and existence of
a similar pathway was observed which creates same morphological changes (Chavez-
Munguia et al., 2007).
Actual reasons behind such transformation of Entamoeba sp. within the host gastro-
intestinal tract is still unknown, though several factors such as high population,
scarcity of nutrients expected to play the key role. To mimic such condition, in vitro
encystation of E. invadens is optimized by using low nutrient and hypertonic medium
termed as LG (Low glucose) medium, (Sanchez et al., 2002) containing approximately
10
47% of the constituents of the normal culture medium (TYIS-33) (Diamond et al.,
1978).
In the course of encystation several morphological changes were observed, like the
clumping of cells, and appearance of cord like structures (Carranza-Rosales et al.,
2000). Mature cyst formation occur within the time period of 60-72 hr, with the
formation of four nuclei along with the fabrication of cyst wall with microfibrillar
appearances surrounding the plasma membrane. The cyst wall was found to be of
different thickness depending on the maturation stage, ranging from 30-50 nm and
120-150 nm. In another study, chitinase filled vesicles are reported in encysting E.
invadens (Ghosh et al., 1999). An ultra structural study of E. invadens has shown the
presence of several vesicles within the cytoplasm, containing electron dense material
similar to the cyst wall composition (Chavez-Munguia et al., 2003). The phenomenon
of multiple vesicle deposition within the cleft between cyst wall and membrane was
also observed. During excystation creation of the operculum may be caused by the
bursting of such vesicles signalled by the environmental stimuli.
Highly insoluble nature of cyst wall material along with tightly packed meshwork is
essential for the survival of the cyst outside the host, and surpasses the water
disinfection system (Erlandsen et al., 1989). Major component of many of the
protozoan cyst wall is expected to be chitin while in a series of studies on Giradia
cyst, filamentous cyst wall is being reported to contain β(1-3) linked N-acetyl
galactosamine polymer, not the chitin (Jarroll et al., 2002). In previous studies
presence of chitin is being documented in the E. invadens cyst (Arroyo-Begovich et
al., 1980 and1982), though a latest study reports the presence of chitosan (deacetylated
chitin) also (Das et al., 2006).
11
degrading enzyme Chitinase was recognized to be important during the encystation
process, as one of its inhibitors was found to inhibit the encystation (Villagomez
Castro et el., 1992) along with the chitin synthase inhibitors (Avron, et al., 1982)
As the 2nd most abundant polysaccharide on earth, chitin was first investigated in 1811
within the cell walls of mushrooms. In 1830 it was also detected in insects and named
as chitin. Chitin is a (1-4)-linked 2-acetamido-2-deoxy-β-D-glucan (Fig. 1.4), and
occur in two different polymorphic forms. These two forms α-Chitin and β-Chitin
differentiate in their packing and polarities of adjacent chains in successive sheets. All
the chains were found to be aligned in parallel manner in β types, while in α type it
occur in anti-parallel manner. In most hydrozoans, nematodes, arthropods, molluscs α-
chitin occur as a major type, while β-chitin occurs in brachiopods, cuttlefish.
Chitin is highly hydrophobic in nature and completely insoluble in water and most of
the organic solvents but solubilises in hexafluoro-isoprapanol, or hexafluoro-acetone.
Solubility of the chitin is remarkably poor due to the high crystallinity supported by
hydrogen bonds between the acetamido groups and depends on the degree of N-
acetylation i.e. the ratio of 2-acetamido-2-deoxy-D-glucopyranose to 2-amino-2-
deoxy-D-glucopyranose (Muzzarelli and Jolles, 1999).
With the advent of several chemical modifications, more soluble polymers are created
from chitin, making it useful for hundreds of application (Muzarelli et al., 1986). Little
more soluble Chitosan was first produced in 1859 belongs to the family of
deacetylated chitins (Fig. 1.4).
12
Commercially chitosan can be produced by removing the acetyl groups by NaOH
treatment. Despite of its deacetylated structure, chitosan still possesses the inherent
crystal structure within, and is sparsely soluble in dilute acids such as acetic or formic
acid. Though most of the natural polysaccharides are neutral or acidic in nature, the
chitin and chitosan form a highly basic group, which is being found to be instrumental
in preparing films, chelate metal ions and polyoxysalt formation.
Along with the soluble counter parts, several other enzymatic products of chitin such
as dimer, trimers were found to exhibit antioxidant activity (Chen et al., 2003), and
other immuno-modulatory effects (Wu et al., 2007) as only di and trimers were able to
pass through the intestinal barrier (Chen et al., 2005).
Biological degradation of chitin into the oligomers, is solely depends on the hydrolytic
enzyme chitinase, which is available in most bacteria, fungal and plant in order to help
in their different endeavours ranging from food digestion to host invasion.
1.9 Chitinases
From the structural point chitinases are classified into two groups, family 18 and
family 19 of glycosyl hydrolase. Family 18 chitinases, contain (β/α)8 catalytic domain,
mainly comprise of bacterial, fungal and mammalian chitinases. This family consists
of more than 200 enzymes, which are further classified by their enzymatic properties.
Most of the family 18 chitinases are well characterized and comprise of single
catalytic domain, along with one or more extra domains at N- or C-terminal end. The
catalytic domain contains several aromatic residues along within the cleft for substrate
binding, with a Glutamine as a conserved amino acid.
13
More than 150 members of family 19 are mostly of plant origin, and not properly
characterized. Difference between these two classes continued from their origin,
sequence, and configuration of hydrolytic cleavage, concluding them as evolutionary
distinct from each other. Other than these two important classes, few minor classes are
also formed, containing chitosanses (family 46).
Chitinases are also characterized depending on their substrate cleavage pattern and
product formation. Chitinase producing fixed length oligomers from any end of the
chitin polymer, classified as exochitinase, while randomly cleaving chitinases are
termed as endochitinase (Deane et al., 1998). Further classification is done to
differentiate the enzymes producing monomers, dimers or oligomers. Evident of
multiple chitinase with different substrate cleavage pattern are observed, and
concluded as instrumental for increased processivity (Horn et al., 2006).
All Entamoeba chitinases belongs to family 18 group and few of them contain the
extra substrate binding domain also. Other than Entamoeba, a large number of
important parasites are reported to produce stage specific chitinase like proteins,
suggesting their importance in those stages (Brydon et al., 1987; Schlein et al., 1991).
Inhibition of such parasitic chitinases either by inhibitor or transmission blocking
vaccine shows promising results in inhibiting parasitic transmission (Shahabuddin et
al., 1993; Dissanayake et al., 1995; Villagomez Castro and Lopez-Romero, 1996).
Chitinase inhibitors may have a potential to act as a pesticide, as the chitinase is being
found throughout the fungal and bacterial kingdom. Allosamidine a
pseudotrisaccharide, obtained from Streptomyces sp. (Sakuda et al., 1986) is found to
be the most potent and efficient inhibitor of both family 18 and 19 of chitinases.
Several other novel chitinase inhibitors such as cyclic peptides (Izumida et al., 1996),
argifin (Shiomi et al., 2000) and argadin (Arai et al., 2000) are being designed and
characterized. Identification of other potential inhibitor by screening drug library may
found to be useful in future (Rao et al., 2005).
One intriguing problem, regarding the usage of parasitic chitinase as a drug target or
vaccine, emerged due to the recent discovery of human chitinases and chitinase like
proteins (Boot et al., 1995 and 1998). Human chitinase and other chitinase like protein
belong to family 18 of glycosyl hydrolase and share an evolutionary common
structure. Though the structural similarity of human and other parasitic chitinases are
14
not that extreme, cross reactivity may occur as an unavoidable result, which can be
circumvented by the studies of structurally unique regions of parasitic chitinases.
Majority of the well known plant and insect chitinases contains single or multiple
domains at either N- or C- terminal end which termed as chitin binding domain
(ChBD) other than the conserved catalytic domain
These domains remain connected to the catalytic domain directly or by means of some
linker peptide. The plant ChBD or the hevein domain is mostly composed of 30-43
residues including eight cysteines, three aromatic residues and glycines (Iwanaga et
al., 1998). Invertebrate chitin-binding domain is being assumed to consist of 65
residues with conserved six cysteine and aromatic residues (Kawabata et al., 1996).
While the invertebrate and plant chitin-binding proteins have similar domain structure
and function, no significant similarity was observed at amino acid level. Different
patterns of disulfide bond formation conclude the absence of any common ancestor for
plant and invertebrate ChBDs. The purpose of the conserved residues among the
insect and plant ChBDs are being evaluated, and it is assumed that all cysteine,
proline, and glycine residues play the structural role, while the conserved polar and
hydrophobic residues are responsible for chitin-binding (Suetake et al., 2000).
Till date, numerous ChBDs are identified and classified as ChBD type I, II and III
(SMART Database) depending on the number of cysteine residues, polar residue and
domain length. The substrate binding completely depends on the aromatic residues,
which are seems to be exposed (Boraston et al., 2004). The reason behind the origin of
these domains is not clear, though in one study it is assumed that, the addition of
ChBD in chitinase occurred only after the separation of chitinases from their
ancestors, as no such domains are observed among lysozymes the near relatives of
chitinases. The reason of this addition is predicted in two possible ways, such as the
gradual evolution from a non-chitin-binding sequence along with the insertion of CBD
from elsewhere in the genome (Shen et al., 1999). The acquisition of a CBD by a
chitinase could increase its activity for insoluble chitin. Number and hydrophobicity
of exposed aromatic residues dictates the substrate binding capacity (Kikkawa et al.,
2008). As well as addition of excess ChBD to any chitinases or subtraction of existing
15
domain drastically changes the enzymatic profile indicating its importance in
insoluble substrate binding (Carmen et al., 2006).
To recognize the potential drug target against E. histolytica, we select the encystation
and excystation specific protein chitinase as a plausible candidate. E. invadens
chitinases are used in this study as a model protein along with E. histolytica chitinase
to identify their role during the encystation. in vitro and in vivo characterization of
these proteins are presumed to be an important task and we focused on the following
objectives in the present study.
16
Chapter 2
For all experimental studies, Entamoeba histolytica (NIH:200 strain) and Entamoeba
invadens (IP1 strain) were used.
Table 2.1: E. coli strains used in this study and their genotypic details
XL1-Blue endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 For expression
(Stratagene) F'[ Tn10 proAB+ lacIq ∆(lacZ)M15] hsdR17(rK- mK+) vector maintenance
[F- = Does not carry the F plasmid, F+ = Carries the F plasmid, F'[ ] = Carries an F
plasmid that has host chromosomal genes on it, Chromosomal genes carried in the F
plasmid are listed in brackets, rB/K+/- = The (B/K) defines the strain lineage, the +/-
indicates whether the strain has or hasn't got the restriction system, mB/K+/- = The
(B/K) defines the strain lineage. The +/- indicates whether the strain has or hasn't got
19
the modification (methylation), hsdS = Both restriction and methylation of certain
sequences are deleted from the strain, hsdR = For efficient transformation of cloned
un-methylated DNA from PCR amplifications, INV = Chromosomal inversion
between locations indicated, ara-14 = Cannot metabolize arabinose, araD = mutation
in L-ribulose-phosphate 4-epimerase blocks arabinose metabolism, ∆( ) =
Chromosomal deletion of genes between the listed genes, deoR and nupG =
Regulatory gene that allows constitutive expression of deoxyribose synthesis genes,
endA1 = For cleaner preparations of DNA due to the elimination of non-specific
digestion by Endonuclease I, (e14) = Excisable prophage like element containing
mcrA gene, galE = Mutations are associated with high competence, increased
resistance to phage P1 infection, and 2-deoxygalactose resistance, glnV = Suppression
of amber (UAG) stop codons by insertion of glutamine; required for some phage
growth, gyrA96 = Mutation in DNA gyrase; conveys nalidixic acid resistance,
lacZ∆M15 = Partial deletion of the lacZ gene that allows α complementation of the β-
galactosidase gene, mcrA = Mutation eliminating restriction of DNA methylated at the
sequence CmCGG (possibly mCG), mcrB = Mutation eliminating restriction of DNA
methylated at the sequence RmC, (φ80) = Cell carries the lambdoid prophage φ80. A
defective version of this phage carrying lacZM15 deletion (as well as wild-type lacI,
lacYA, and flanking sequences) is present in some strains, recA1 and recA+ = For
reduced occurrence of unwanted recombination in cloned DNA; cells UV sensitive,
deficient in DNA repair, relA = Relaxed phenotype; permits RNA synthesis in absence
of protein synthesis, Tn10 = Transposon normally carrying Tetracycline resistance,
Tn5 = Transposon normally carrying Kanamycin resistance]
2.1.3 Primers
For cloning and RT-PCR purpose primers were designed using Oligo Perfect primer
designing software (Invitrogen). During primer designing Tm was determined
depending on the primer length and GC content. Restriction enzyme sites and ATG
sequences were added depending on the requirement. All the primers were obtained in
de-salted condition.
20
2.1.4 Molecular Biology Materials
100 bp marker from NEB contains fragments of 1517, 1200, 1000, 900, 800, 700, 600,
500, 400, 300, 200, 100 bp.
1 kb marker from NEB contains fragments of 10,000, 8000, 6000, 5000, 4000, 3000,
2000, 1500, 1000, 500 bp.
1 kb plus marker from Invitrogen, contains bands ranging from 500 bp to 12 kb.
The PMWM (Medium) range from Bangalore Genei contain these following bands of
97.4, 66.0, 43.0, 29.0, 20.0, 14.3 kDa. The Fermentas unstained marker range contains
proteins of 116, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4 kDa. The pre-stained marker from
Fermentas contains band of 129.0, 85.0, 50, 36, 28 and 21 kDa. Kaleidoscope pre-
stained marker obtained from Bio-Rad contains bands of 250, 150, 100, 75, 50, 37, 25,
20, 15 and 10 kDa.
For Immunodetection studies, Anti Rabbit IgG conjugated with TRITC, Phalloidain-
FITC, DAPI, Sytox Green, FITC were obtained from Sigma and Molecular Probes.
21
Anti rabbit IgG conjugated with HRP and Protein A-HRP for western blot were
obtained from Sigma and Bangalore Genei respectively.
2.1.7 Chemicals
All Polymerase Chain Reactions were done in Perkin Elmer 2400 series or Applied
Biosystems Thermocycler. Ligation was done using Eppendorf thermostat ligation
bath. Centrifugations were done using Eppendorf refrigerated and non-refrigerated
centrifugation system with fixed angle rotor and swing bucket rotor as per the
requirement. All microbiological work and cell culture work were done in BioKleanzd
Biosafety Laminar hood. For sonication purpose pulse sonicator of Mysonix was used.
Agarose and polyacrylamide Gel electrophoresis, Western blotting, electro-elution
were performed with Tarson, GE-Amersham, and Bio-Rad apparatus.
Enzymatic assays were done in SPEX Fluromax-3 of Horiba Zobin Vyon and Varion
Spectroflurometer. HPLC analysis was done in Agilent 1100 series. DNA
visualization in agarose gel was done by UV Transilluminator from Bioview,
Germany. DNA and Protein concentration were checked in Nanodrop
22
spectrophotometer, UV-Visible Spectrophotometer of Beckman Coulter and Varian.
For pipetting, Pipetman from Tarson, Aqupippete from Gilson and Steripipette from
Erba BioHit were used. Sterilized and filter tips were obtained from Tarson and
Axygen. Microcentrifuge tubes of 0.2, 0.5, 1.5 and 2 ml were brought from Tarson.
All the glass wares were obtained from Borosil and plastic wares brought from Tarson.
Bacterial culture and Entamoeba cultures were incubated in New Brunswick Shaker-
incubator and Beckman Coulter incubator respectively. All the Entamoeba cell culture
was maintained in 50 ml Nunc cell culture flask and 15 ml glass culture tube of
Borosil. Bright field Microscopy of Entamoeba cells was done with Leica S4E and
Olympus CKX31. For fluorescence microscopy Leica MPS 60 and Olympus IX51
were used. For confocal microscopy Olympus Flow view FV1000 was used, with
argon and HeNe laser.
LB medium was prepared as mentioned above and 1.5% agar was added before
autoclaving. After autoclaving medium was cooled to 55oC and antibiotics were added
to the medium. Solidified plates were stored at 4oC for future use.
Antibiotic solution
DNA buffers
24
Preparation of 0.5 M EDTA (pH 8.0)
EDTA of 18.61 gm was added to 70 ml of sterile water. The solution was stirred
vigorously in a magnetic stirrer and NaOH pellets were added to adjust the pH of the
solution to 8.0. Approximately 8 gm of NaOH pellet required to dissolve the EDTA
completely. The volume was adjusted to 100 ml, and the solution was autoclaved.
To make 1 liter of 10% SDS (pH 7.2), 100 gm of electrophoresis grade SDS was
dissolved in 900 ml of water. The solution was heated at 68oC to dissolve the SDS
completely. The pH of the solution was adjusted to 7.2 by adding a few drops of
concentrated HCl and then volume was adjusted to 1 liter. The solution was then
dispensed into aliquots.
Bromophenol blue (0.25%) and glycerol (30%) was dissolved in 10 ml of water and
stored at 4°C.
25
Tris saturated Phenol
The liquid phenol was then extracted several times by vigorous shaking at room
temperature with equal volumes of 1.0 M Tris-HCl (pH 8.0) followed by 0.1 M Tris-
HCl (pH 8.0) at room temperature. The aqueous layer was removed carefully and the
extraction procedure continued until the pH of the aqueous phase was >7.6. Extracted
phenol can be stored at 4°C at 0.1 M Tris-HCl or equilibration buffer for months.
X-gal
Amorphous form of EtBr of 1 g was dissolved in 100 ml of sterile water and stored in
room temperature.
Preparation of solution I
To make solution I, 25 mM Tris-base (pH 8.0) was mixed with 10 mM EDTA (pH 8).
26
The volume was made up to 100 ml with sterile water. The solution was autoclaved
for 15 minutes at 10 psi and stored at 4oC.
Preparation of solution II
Solution II was prepared fresh every time prior to plasmid DNA isolation. To make
fresh solution II, 0.2 N NaOH (freshly diluted from 10 N stocks) was added to 1%
SDS (from 20% SDS stock). The volume made with distilled water and mixed
thoroughly several times.
Protein buffers
Tris-HCl (pH 6.8) of 1 M and 0.4% of SDS was dissolved and volume adjusted with
water. The solution was stored at 4°C.
27
10% ammonium persulfate
TEMED
TEMED was obtained commercially from Sigma as liquid and stored at 4°C.
Tris base of 3 gm (25 mM), 14.4 gm of glycine (192 mM) and 1gm SDS (0.1%)
dissolved in water and volume made up to 1 liter. pH was found to be 8.3 without any
adjustment.
5X Sample buffer
To prepare 1 liter destain solution, 100 ml methanol dissolved in 100 ml glacial acetic
acid and 800 ml water and stored in dark bottle at room temperature.
Electrodialysis buffer
Ammonium hydrogen carbonate and SDS was dissolved in 1liter of distilled water to
the final concentration of 15 mM and 0.025% (w/v) respectively.
28
Silver staining buffer
Fixing solution
Methanol and formalin of 40% and 13.5% of final volume was mixed and final
volume was made 250 ml by adding distilled water.
Sodium thiosulphate of 0.05 gm was dissolved in 250 ml distilled water to make the
final stock of 0.02%.
Silver nitrate of 0.25 gm was dissolved in 250 ml water to make the final stock 0.1%.
Developing solution
Stopping solution
To prepare 1X PBS solution, 8 gm of NaCl (137 mM), 0.2 gm of KCl (2.7 mM), 1.44
gm of Na2HPO4 (dibasic anhydrous) (10 mM), and 0.24 gm of KH2PO4 (monobasic
anhydrous) (2 mM) were mixed in 900 ml of water. Final pH was adjusted to 7.2 with
HCl and then final volume of water was adjusted to 1 liter. The solution was
autoclaved and stored at room temperature.
All the buffers were prepared fresh and used in chilled condition.
29
Equilibration buffer
Tris base of 3gm and 14.4 gm of Glycine were dissolved in distilled water and the
final volume was made up to 1000 ml.
Transfer buffer
To 1000 ml of equilibration buffer 200 ml methanol was added and the buffer was
kept in 4°C till further use.
Blocking buffer
Polyclonal or monoclonal Primary antibody was raised in rabbit or brought from the
supplier respectively and diluted in 1X PBS at 1:1000 ratio.
Protein A-HRP or Anti Rabbit IgG conjugated with HRP was obtained from the
supplier and diluted in 1X PBS at 1:1500 ratio.
Detection solution
Immunostains
4% PFA
DAPI
To make a 5 mg/mL stock of DAPI, contents of one vial (10 mg) was dissolved in 2
ml of deionized water or dimethylformamide. Aliquots of 200 µl was prepared and
stored in -20°C
Stock solutions of Phalloidin conjugates have been made in methanol or DMSO at 0.1
to 5 mg/ml. Aliquots was prepared and kept in -20°C. Final dilutions made in aqueous
physiological buffers.
Stock solutions was made 1–5 mg/ml in 0.1 M sodium bicarbonate (approximate pH
8.3) and stored in -20°C in aliquots.
Polyclonal anti EiChit2 antibody was raised in a rabbit following the protocol of 3
boosters and 3 collections. After final booster, 15 ml blood was collected from the
heart of the rabbit and the serum was stored in -20°C.
Anti rabbit IgG solution was kept in 2-8°C and diluted at 1:500 times in 1X PBS.
31
200 mM Sodium phosphate buffer (pH 6-8)
Sodium acetate trihydrate and glacial acetic acid were dissolved in water to obtain
different pH after pKa calculation.
Lysis buffer for protein purification under native condition (pH 8.0)
Lysis buffer or sonication buffer was prepared by adding 6.9 gm of NaH2PO4.H2O (50
mM), 17.54 gm NaCl (300 mM), and 0.68 gm imidazole (10 mM) in double distilled
water. The solution pH was adjusted to 8.0 with 10N NaOH.
The solution was consisting of 6 M Guanidium hydrochloride and 0.2 M acetic acid
dissolved in required amount of sterile water. The solution was stored at room
temperature.
32
Sodium azide (0.02%)
Sodium azide of 20 mg was dissolved in 100 ml of sterile water. The solution was
used for storing columns.
1 M Dithiothreitol (DTT)
100 mM IPTG
Amorphous saliter of IPTG (mol. wt. 238.3 g/mole) of 238 mg was dissolved in 10 ml
of sterile water and sterilized by 0.22 µm disposable filter. Aliquots of 1 ml are made
and stored at -20°C. Final concentration for induction was used as 1 mM or lower than
that.
Colloidal chitin
Chitin powder was obtained from the suppliers, and incubated with concentrated HCL
at 4°C for overnight with continuous stirring. The supernatant was mixed drop-wise
with 50% chilled ethanol. The supernatant was washed vigorously with distilled water
until the pH became neutral. The colloidal material was freeze dried and stored.
33
Entamoeba Cell culture medium
Triple filtered adult bovine serum was obtained from the suppliers. Heat inactivation
was done by incubating the serum in 65°C for 30 min. The serum was then cooled and
kept in 4°C in aliquots. To prepare 1000 ml of TYI broth, Bacto peptone of 30 gm,
Glucose of 10 gm, NaCl of 2gm, L-Cysteine Hydrochloride of 1gm, Di-Potassium
hydrogen phosphate of 1gm, Potassium Di-hydrogen phosphate of 600 mg, Ascorbic
acid of 200 mg and 1 ml of Ferric ammonium citrate (22.8 mg/ml) were dissolved in
500 ml of double distilled water and pH was adjusted to 6.8 using 10N NaOH. Final
volume was made up to 870 ml and sterilized by autoclaving for 15 min. The
incomplete medium was used for preparing complete medium, and stored in 4°C.
Different compositions named as Sol I, Sol II, Sol III, Sol IV and Sol V were prepared.
34
Part II was prepared by mixing 12.5 mg of Niacinamide, 12.5 mg of Pyridoxine-HCl,
12.5 mg of Pyridoxal-HCl, 5 mg of Thiamine-HCl, 5 mg of Ca-pantothenate, 25 mg of
Myo-inositol, and 250 mg of Choline chloride to double distilled water and the final
volume made up to 25 ml.
Sol II- 10 mg of D-Biotin and 1ml of 1N HCl was added to double distilled water and
the final volume was made to 100 ml.
Sol III- 10 mg of Folic acid was added to 100 ml of distilled water (80°C).
Stock solutions of sol I, sol II, sol III, sol IV, and sol V were prepared at first and
added in following amount to prepare the NCTC mixture, Sol I: Sol II: Sol III: Sol IV:
Sol V:: 2: 1: 1: 10: 1 part.
Encystation medium
35
Cryopreservation solution
Axenic culture medium base (TYI) and serum (heat inactivated bovine serum) were
mixed in same volume (1:1) to prepare the Solution I. Cell culture grade DMSO of
0.75 ml was mixed to 5 ml of this solution I, to reach the final concentration of 15%
and the mixture was termed as solution II.
2.1.10 Kits
Genomic DNA isolation kit was obtained from Promega (USA) and used for
Entamoeba genomic DNA isolation. Total RNA isolation kit or TRI Reagent solution
was obtained from Ambion (USA). Plasmid DNA miniprep kit was obtained from
Promega. QIA quick Gel extraction kit was brought from Qiagen (Germany).
Retroscript or the cDNA synthesis kit was purchased from Ambion (USA).
2.1.11 Vectors
TA vector was used to facilitate one step cloning after PCR amplification. Blue-white
selection was used for preliminary screening of recombinant clones.
Vectors from two different sources were used in this study and their genetic structure
and specificity were described below.
pGEM-T system is very convenient for one step PCR product cloning. Originated
from pGEM-5Zf (Promega, USA), this vector contains one 3’ terminal thymidine to
both ends (Fig. 2.1).
pGEM-T vector contains T7 and SP6 RNA polymerase promoters flanking MCS,
within the α peptide-coding region of the enzyme β-galactosidase, and the origin of
replication of the filamentous phage f1. Insertional inactivation of α-peptide allows
colour screening of recombinant clones. The pGEM-T vector contains multiple
restriction sites within the MCS for easy release of cloned fragment. The vector is of 3
kb molecular weight, and ampicilline resistant gene was incorporated as antibiotic
marker.
36
Fig. 2.1: Schematic diagram and sequence map of pGEM-T vector (Promega manual).
This vector represents the same basic mechanism and facility for one step PCR
product cloning (Fig. 2.2).
Fig 2.2: Schematic diagram and sequence map of pTZ57R/T vector (Fermentas manual)
37
Commercially obtained from Fermentas, this plasmid is informally known as pFERM.
The vector is of 2.8 kb, and contains f1 replication origin. The MCS is flanked by α-
peptide coding region and T7 promoter. β-Lactamase gene (bla) confers the
ampicilline resistance. The linear vector contains two 3’ thymidine at the open ends
for insert binding. Multiple restriction sites are available within MCS for insert
release. Blue-white screening was used for preliminary clone identification.
Over expression of heterologous protein within E. coli system generally done by pQE
vector system commercially obtained from Qiagen (Fig.2.3). 6xHis-tagged protein was
over expressed using T5 promoter transcription-translational system.
Fig.2.3: Schematic diagram and sequence map of pQE30 vector (Qiagen Handbook)
Primarily originated from pDS family of plasmids, this group of vector emerges after
several derivations and weights 3.4 kb. Phage T5 promoter and two lac operator
ensure efficient repression of expression in absence of induction, and resulted in
strongly controlled expression. 6xHis-tag coding sequence attached to N terminal of
cloned sequence. MCS and translational stop codons were available in all reading
frames, flanked by several restriction sites. Origin of replication belongs to ColE1, and
β-lactamase gene confers the ampicillin resistance. Strong ribosomal binding site
38
ensure high translation rate, and dual transcriptional terminator ensure the expression
stability.
All microbiological methods were done by following Sambrook and Russell (2001) if
not stated otherwise.
For the transformation purpose competent cells were prepared by using calcium
chloride method. Overnight grown bacterial culture of 200 µl was used as innoculum
to 20 ml of native LB and incubated for 3-4 hr at 37°C to reach the logarithmic phase.
After 3-4 hr, the O.D of the culture media reached 0.5-0.6, which indicates the log
phase of bacterial growth. The bacterial solution was kept in ice for 5 min and then
centrifuged at 5000 r.p.m. for 5 min at 4oC. After the centrifugation, the supernatant
was removed and the bacterial pellet was suspended in 3 ml of ice cold 100 mM
calcium chloride and kept in ice for 45 min. Exposure to calcium ions enables the cells
to uptake DNA or to become “competent”. Thereafter centrifugation was done again at
5000 r.p.m. for 5 min at 4°C. Supernatant was discarded and bacterial pellet re-
suspended in 1 ml of 100 mM calcium chloride solution. Dissolution of the pellet
slowly gives rise to competent cell.
For long time storage, the cells were dissolved in 1 ml of 100 mM cold calcium
chloride: glycerol (85:15) solution. Suspensions of 50 µl were stored in sterile
microcentrifuge tubes, flash frozen in liquid nitrogen and stored at -70oC for future
use. Such frozen cells retain their competency for months.
39
2.2.3 Transformation
The rationale behind the blue white screening is the disruption of the lacZ gene
responsible for the β-galactosidase enzyme and unavailability of the enzyme for X-gal
degradation. With the cloning of insert in the MCS site, the gene gets disrupted and
the absence of β-galactosidase enzyme resulted in the white colonies on the X-gal
coated plate, where the non-recombinant clones produce the enzyme and degrade the
substrate forming blue colored colonies. The white colonies were picked up and grown
in 3 ml LB medium supplemented with 100 µg/ml of ampicillin or required
antibiotics.
Once the right clone was detected, glycerol stock was made for long term storage.
Single colony was isolated and grown for overnight in 1 ml of LB medium containing
required antibiotic at 37°C with vigorous shaking at 250 rpm. 800 µl of such overnight
grown culture was mixed with 200 µl of sterile glycerol (20% v/v) and vortexed
briefly to mix the contents well. The cryovials were flash frozen in liquid nitrogen and
stored at -70oC for future use.
40
2.3 Molecular Biology methods
All molecular biology methods were done by following Sambrook and Russell (2001)
if not stated otherwise.
50 ml culture flasks with log phase Entamoeba cells were chilled with ice and cells
were harvested and transferred in to 1.5 ml microcentrifuge tube. Cells were pelleted
down by centrifugation at 1500 r.p.m. for 3 min. The genomic DNA was isolated
following the manufacturer’s protocol. Briefly the supernatant were removed and 200
µl PBS was added to wash the cells. The cells were re-suspended in 1xPBS followed
by the addition of 600 µl Nuclei Lysis solution to lyses the cells. 3 µl of RNase
Solution was added to the mixture and the sample was mixed by inverting the tube 2-3
times. The mixture was incubated for 15-30 min at 37°C. The sample was cooled to
room temperature for 5 min before proceeding further. To the mixture, 200 µl of
Protein precipitation solution was added and vortex vigorously at high speed for 20 s.
The sample was chilled on ice for 5 min. The mixture was centrifuged for 4 min at
13,000×g. The precipitated protein forms a tight white pellet. The supernatant
containing the DNA was removed carefully and transfer it to a clean 1.5 ml
microcentrifuge tube. 600 µl of isopropanol was added to the solution and gently
mixed by inversion until the white thread-like strands of DNA form a visible mass.
The tube was centrifuged for 1 min at 13,000×g at room temperature. The DNA was
precipitated as small white pellet. The supernatant was decanted carefully and washed
with 600 µl of 70% ethanol. The tube was centrifuged for 1 min at 13,000×g at room
temperature. The ethanol was carefully aspirated and the DNA pellet was air-dried for
10-15min. 100 µl of DNA rehydration Solution was added to rehydrate the DNA by
incubating at 65°C for 1 hr. The DNA was stored at 2-8°C.
The culture flasks containing Entamoeba cells were chilled by incubating them in ice,
and the cells were pelleted down, by centrifugation at 1500 r.p.m. for 3 min. The total
RNA was isolated following the manufacturer’s protocol. Briefly 1 ml of TRI Reagent
41
solution per 5-10 x 106 cells was added and incubated for 5 min at room temperature.
The mixture was centrifuged at 12,000x g for 10 min at 4°C and the supernatant was
transferred to a fresh microcentrifuge tube. 200 µl of chloroform per 1 ml of TRI
Reagent solution was added, mixed well, and incubated at room temp for 5-15 min.
The tube was centrifuged at 12,000 x g for 10-15 min at 4°C, and the aqueous phase
was transferred to a fresh tube. 500 µl of isopropanol per 1 ml of TRI Reagent solution
was added to the aqueous solution and mixed well for 5-10 s, followed by incubation
at room temp for 5-10 min. The tube was centrifuged at 12,000 x g for 8 min at 4-
25°C, and the supernatant was discarded. 1 ml of 75% ethanol per 1 ml of TRI
Reagent solution was used to wash the pellet. The ethanol was washed away by
centrifugation at 7,500 x g for 5 min. The RNA pellet was briefly air dried and
dissolved in nuclease free water. Isolated RNA was stored in -20°C.
Small scale and large scale plasmid isolation was done by alkaline lysis method using
the standard protocol. For small scale isolation of plasmid DNA, 3 ml of full grown
bacterial culture was used. The cells were grown overnight for 16 hrs at 37oC in L.B
medium supplemented with appropriate antibiotics at required concentrations at 250
r.p.m. The cells were pelleted down by centrifugation at 5000 r.p.m. for 5 min.
Supernatant was removed completely and the bacterial pellets were re-suspended
completely in 200 µl of cold solution I. The cells were then vortex well to loosen the
cell pellet so that each and every cell gets exposed to lysis solution. 400 µl of freshly
prepared solution II was added. SDS from sol II solubilize the phospholipid and
protein components of cell membrane leading to cell lysis and release cell components
and denatures the chromosomal and plasmid DNA. The solution was mixed properly
by inverting the microcentrifuge tube slowly (to avoid shearing of chromosomal
DNA) and kept in ice for exactly 5 min which allows maximal release of plasmid
DNA from the cell. The cell lysate was then neutralized by the addition of 300 µl of
solution III and the tubes were inverted slowly several times and kept in ice for 10
min. Here the high salt content precipitates the denatured protein, chromosomal DNA
and cell debris forming insoluble salt-detergent complex. Plasmid DNA being circular
and covalently closed renatures quickly and remains in solution.
42
To separate out the plasmid DNA, the tubes were centrifuged at high speed of 12,000
r.p.m. for 10 min. Supernatant was saved in a new sterile microcentrifuge tube and
equal volume of isopropanol was added. The microcentrifuge tube was allowed to
stand in room temperature for 20 min followed by centrifugation at 10,000 r.p.m. for
10 min. Supernatant was discarded and 200 µl of 70% cold ethanol was added to wash
the pelleted plasmid DNA. Ethanol precipitates out intact nucleic acid molecules. The
residual ethanol was removed by brief centrifugation, pellet was air dried and then
dissolved in required volume of sterile water. RNaseA (5mg/ml) was added to the
solution to a final concentration of 20 µg/ml and the solution was incubated at 37°C in
a water bath for 30 min.
Equal volumes of Tris saturated phenol: chloroform (1:1 v/v) was added to the RNase
treated solution, thoroughly mixed by vortexing and centrifuged at 10,000 r.p.m. for
10 min. Phenol being denser than water remains below where the nucleic acids remain
in the upper aqueous layer. After centrifugation the upper aqueous layer was slowly
pipetted out into a sterile microcentrifuge tube, without disturbing the double layer of
phenol and aqueous material. The same procedure was repeated again with equal
volume of chloroform and the upper aqueous layer was collected. The aqueous layer
containing the DNA was precipitated out with 1/10 volume of 3 M Sodium-acetate
and 2.5 times of dehydrated alcohol. The solution was kept at -20°C and then
centrifuged at 10,000 r.p.m. for 10 min to pellet plasmid DNA. Supernatant was
discarded and 200 µl of 70% cold ethanol was used to wash pelleted DNA. The
residual ethanol was removed by brief centrifugation.
The DNA pellet was air dried completely to make it absolutely ethanol free. Then
plasmid was dissolved in required amount of autoclaved distilled water. For large
scale isolation of plasmid DNA, cells were harvested from 250 ml LB/YT medium
supplemented with required antibiotics at 37°C following the same procedure with
scaled up solution.
From the isolated total RNA 1-2 µg was used for cDNA synthesis by following the
manufacturer’s protocol. Briefly in a typical reaction of 20 µl, sequentially 2 µl Oligo
43
(dT), 2 µl 10X RT Buffer, 4 µl dNTP mix, 1 µl Placental RNase Inhibitor, 1 µl
Reverse Transcriptase were added. The final volume was made up to 20 µl by adding
Nuclease-free Water. The mixture was mixed gently centrifuged briefly and incubated
at 44°C for 1 hr followed by 92°C for 10 min to inactivate the Reverse Transcriptase.
The reaction mixture was stored at -20°C before proceeding to the PCR.
2.3.5 RT-PCR
RT-PCR was carried out by using gene specific primers designed to amplify a small
region (~450bp) of the gene of interest. A typical RT-PCR mixture of 50 µl was
prepared containing 1-5 µl RT reaction mixture, 5 µl 10X PCR buffer, 2.5 µl dNTP
mix, 2.5 µl PCR primers, 1 unit Taq DNA Polymerase and nuclease free water. The
reaction was carried out for a cycle of 94°C for 2- 4 min, 30 cycles at 94°C for 30 s,
55°C for 30 s, 72°C for 40 s and then by a final extension at 72°C for 5 min. Positive
and negative controls were done by using Actin template and cDNA mixture
respectively.
PCR amplification of gene of interest was completed by using gene specific primers.
For cloning purpose Hi-fidelity Expand Taq polymerase was used, while Taq
polymerase was used otherwise.
In a typical reaction of 50 µl, 20 ng of template DNA, 0.5 unit of Taq polymerase, 0.5
µl of dNTP mixture (100X stock solution; 10 mM of each), 20 pmole of sense and
antisense primers, 5 µl of Hi fidelity Expand buffer or PCR buffer (10X stock
solution) were used, and the rest of the volume was made up to by adding autoclaved
PCR water. The whole PCR reaction was carried out for a cycle of 95°C for 5 min, 35
cycles at 95°C for 30 s, 55°C for 30 s, 68°C for 60 s (72°C for Taq) (60 s for every
1000 bp long fragment) and then by a final extension at 72°C for 5min.
44
In a standard reaction mixture of 100 µl containing 10 µg of plasmid DNA, 1 µl of
required enzymes were added (1unit/100 µl). Required buffer (10X stock) of 10 µl and
BSA (100X stock) of 1 µl (if suggested) were added. The final volume was made up
by autoclaved water. Both double and sequential digestions were carried out by
incubating the reaction mixture for 3 hr at 37°C water bath. In the situation of double
digestion, the buffer and enzymes of previous digestion were removed by alcohol
precipitation. After the completion of first digestion 1/10 times ice cold 3 M sodium
acetate and 2.5 times of dehydrated alcohol was added to the reaction mixture. After
20 min incubation at -20°C, the reaction mixture was centrifuged at 10,000 r.p.m. for
10 min. Further washing was done by adding 200 µl ice cold ethanol and
centrifugation for 10 min at 10,000 r.p.m. The pelleted DNA was air dried and
dissolved in distilled water and treated further with the next restriction digestion
enzyme, with high salt containing buffer.
To visualize the isolated DNA or RNA, agarose gel electrophoresis was done which
helps to separate the oligonucleotide fragments. To cast 0.8% agarose gel, 0.4 g of
molecular grade agarose was dissolved in 50 ml water containing 0.01%
electrophoresis buffer. The agarose was then cooled down to 60°C, and Ethidium
bromide was added to a final concentration of 0.5 µg/ml. The melted agarose solution
was poured in the pre-assembled electrophoresis tray with the comb in proper position
and the gel was allowed to solidify. After solidification, the comb was removed
carefully and the gel was mounted in an electrophoresis tank containing enough 1X
TAE buffer to cover the gel completely. The DNA/RNA samples were mixed well
with the 6X loading dye and then slowly loaded in to the slots of the submerged gel
using micropipettes. The gel was run at 70 volt until the bromophenol blue dye
migrated to about 1 cm from the bottom of the gel. The ethidium bromide stained gel
was then observed using an UV Transilluminator.
To elute DNA from agarose gel QIA quick Gel Extraction kit (QIAGEN) was used.
45
The kit also used to cleans up DNA from enzymatic reactions. After the gel
electrophoresis, DNA fragment was excised from the agarose gel with a sterile sharp
blade. The gel piece containing the DNA was weighed in clean microcentrifuge tube
and thrice volume of QG buffer was added per amount of gel weight (100 mg of
gel~300 µl). The solution was incubated at 50°C for 10 min and vortex vigorously to
dissolve the gel completely.
The QIA quick spin column was placed in a 2 ml collection tube and centrifuged at
10,000 r.p.m. for 1 min, which allows binding of the DNA to the membrane. Flow
through was discarded and 750 µl of ethanol mixed PE buffer was added to QIA quick
column and centrifuged at 10,000 r.p.m. for 1 min. This PE buffer washes the
membrane. Flow through was discarded and again centrifuged for an additional 1 min
at 10,000 r.p.m. to remove residual PE buffer.
The QIA quick column was placed in a fresh 1.5 ml microcentrifuge tube. To elute the
DNA 30 µl of autoclaved distilled water was added to the QIA quick column and then
centrifuged at 10,000 r.p.m. for 1 min. The eluted DNA was collected in the
microcentrifuge tube and it was checked by agarose gel electrophoresis.
For protein and RNase free DNA the ratio of A260/A280 should be close to 2. RNase
contamination will increase the ratio.
46
For estimating RNA, concentration was estimated assuming that an O.D260nm of 1
corresponds to 40 µg/µL of RNA. Thus the formula stands: O.D260nm x 200 [dilution
factor] x 40 µg/ml = RNA µg/ µl.
2.3.11 Ligation
For ligation with pTZ57R/T or pGEM-T, the ligation mixtures were prepared by
following the manufacture’s protocol. For ligation of the pQE30 vector and insert
molecule, the 1:5 molar ratio of vector to insert was used. With that respect the
concentration of vector and insert was taken in the ligation mix. A typical ligation
mixture contains 1 unit T4 DNA Ligase enzyme (1unit/1µl), 1µl Ligase buffer, vector
and insert to make up the final volume up to 10 µl. The reaction mixture was
incubated at 16°C for 16 hr.
To check the fidelity of the sequences, sequencing reaction was done using Applied
Biosystems automated DNA Sequencer. Sequencing reaction was consist of 300 ng of
purified plasmid DNA, 2.0 µl of 2.0 pico mole primers, 2.0 µl of Big DyeTM
Terminator Cycle Sequencing Ready Reaction Mix (Applied Biosystems, USA) and
sterile double distilled water to a final volume of 10 µl prepared in sterile 0.2 ml tubes.
All the additions were carried out in ice. Thermal cycling was carried out following
the ABI recommended cycling proge of 96°C for 30s, 50°C for 15 min and 60°C for 4
min. The sequence products were purified using ABI recommended ethanol
Tm
precipitation for Big Dye Terminators by adding 60 µl of 100 % ethanol to the 10
µl sequencing reaction volume. The solution remained at room temperature for a
minimum of 15 min before centrifuging at 3000 r.p.m. for 45 min using swing bucket
rotor system of refrigerated centrifuge. The supernatant was discarded by inverting the
plates followed by short centrifugation at 1000 r.p.m. for 1 min. The DNA pellet was
washed with 70% cold ethanol, briefly centrifuged and the remaining supernatant was
removed by pipette tip. The sequencing tubes were stored in dark at 4°C prior to use.
The purified DNA samples were re-susupended in 12 µl of Hi-Di-Formamide and
47
were denatured at 96°C for 5 min. The samples were then loaded to the Sequencer for
reading.
Both the growth of expression culture and purification of the protein were done
following the Ni-NTA Spin Handbook (Qiagen) and Protein methods (Bolleg et al.,
1996). The principle is based on IMAC principle (Immobilized Metal Affinity
Chromatography) where the poly-Histidine tag acts as the metal chelating group. The
pQE-30 expression plasmid consists of a full-length gene cloned between the BamHI
and SalI cloning sites of the expression plasmid. The gene is fused in-frame with N-
terminal peptide containing six tandem Histidine residues, and expression of the His-
tag protein is driven by a T7 RNA polymerase promoter.
Recombinant pQE30 plasmid was transformed into E. coli M15 and plated on
Amp/Kana LB agar plates. Colonies were picked and inoculated in 3 ml culture for
overnight incubation at 37°C and shaking at 250 r.p.m. 25 ml of LB media was
inoculated further for protein expression study and shake vigorously at 37°C until the
O.D600nm reaches between 0.5-0.7. The recombinant protein expression was induced by
adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture and the
incubation continued at 37°C for 5 hr. After the incubation period, cells were harvested
by centrifugation at 5000 r.p.m. for 6 min and the supernatant was discarded. Bacterial
pellet were then washed and re-suspended in 2.5 ml of 1X PBS (pH 7.2) containing 1
mM PMFS and 1 mM DTT. Cells were sonicated by using ultrasonic cell disrupter, for
15 min with 30s pulse on ice. The clear solution was centrifuged at 13,000 r.p.m. for
45 min at 4°C to precipitate out the cell debris and impurities. The supernatant was
stored separately and the pellet was washed thoroughly with 1X PBS. The pellet was
dissolved in same amount of 1X PBS and vortex briefly. To detect the solubility of the
recombinant expressed protein, samples were aliquoted from supernatant and the
pellet fraction, treated for SDS-PAGE analysis.
48
2.4.2 SDS-PAGE
For SDS-PAGE, the method of Laemmeli was followed. Bio-Rad gel apparatus was
used for gel casting. The glass plates of mini gel apparatus were cleaned and
assembled properly on a gel caster.
The ingredients Solution A (4 ml), Solution B (2.5 ml), Water (3.5 ml), 10% APS (50
µl) and TEMED (5 µl) were mixed to cast 10 ml of 12% resolving gel.
After 15 min, 5% stacking gel of 5 ml was casted using the following components
such as Solution A (0.67 ml), Solution C (1 ml), Water (2.3 ml), 10% APS (30 µl) and
TEMED (5 µl) above the resolving gel.
The comb was inserted in proper position. After 30 min of polymerization, the comb
was removed carefully and the wells were washed properly with 1X running buffer to
remove un-polymerized acrylamide. Gel was fitted into the electrophoresis tank and
1X Tris glycine electrophoresis buffer was poured into the tank to cover the vertical
glass plates. 20 µl of protein samples were boiled with 4 µl of 5X sample buffer for 3
min at 100°C, and loaded into the wells. The gel was run at 70 Volt till the protein
sample entered the resolving gel after crossing the stacking gel. Then the gel was run
at 100 Volt till the dye font is 1 cm away from the end. After completion of the run gel
apparatus was dismantled carefully and the gel was taken out. Detection of migrated
bands was done either by Coomassie blue staining or by silver staining.
Coomassie brilliant blue R-250 binds nonspecifically to almost all proteins, which
allows detection of protein bands in polyacrylamide gels. Gels are soaked in 1X
staining solution with gentle shaking in rocker for 1 hr or for longer time at room
temperature. After that gels are washed with 1X Destain solution with several changes
till the bands were visualized in naked eye.
Protein bands in SDS-PAGE gel containing less than microgram of protein were not
visible by Coomassie blue staining. Silver staining technique was done to detect nano
49
gram level of protein. The gel was soaked for 10 min in fixing solution. Washing of
the gel was done twice by water for 5 min each. Impregnation of the gel was done by
soaking it in 0.02% Na2S2O3 for 1 min. Further washing was done twice by water for
20 s. Silver staining was done by immersing the gel in 0.1% AgNO3 for 10 min. The
stained gel was rinsed with water and again with a small volume of developing
solution. For development of the stain, the gel was soaked in fresh developing solution
until band intensities are adequate (~1-3 min). 12.5 ml of 2.3 M citric acid was added
to stop the development and incubated for 10 min, followed by the washing for 10 min
in water. The developed gel was then soaked in water for 30 min or longer before
drying.
After the expression profile was analyzed, protein purification was done utilizing the 6
Histidine tag at the N-terminal end of the protein sequence. The cytosolic protein was
purified following the suppliers protocol. Ni-NTA agarose beads were obtained from
Qiagen. Slurry (5%) of 1 ml volume was loaded on a glass column and washed with
water followed by the equilibration with water or 1X PBS. After the beads were
settled, the supernatant was applied on the column and the flow through was collected.
To minimize the non-specific bindings, the column was washed several times with the
wash buffer (pH-8). Washing was continued till O.D280nm reaches less than 0.1. Bound
proteins were eluted stepwise with elution buffer containing gradient of 50, 100, 200
and 500 mM imidazole. The elution was monitored by watching the O.D280nm. Initially
all the fractions were collected and stored at 4°C for further analysis by SDS-PAGE.
Single column was used for at least 3-4 times for a single protein. To regenerate the
leached Ni ions, following steps were done by following the manufacturer’s protocol.
Used columns were washed with 2 column volumes of Regeneration Buffer, followed
by the wash with 5 volumes of water. 2% SDS solution of 3 volumes were used to
solubilise the excess protein, which was further gradually washed by sequential
addition of 1 volume of 25%, 50% and 75% ethanol, followed by 5 volumes of 100%
Ethanol. Further rehydration of column was done by washing with 1 volume of 75%,
50
50%, and 25% Ethanol, followed by 1 volume of water. Finally the column was
washed with 5 volumes of 100 mM EDTA (pH 8.0). Enough water was used to wash
away the residual alcohol and debris. The column was then replenished with 2
volumes of 100 mM NiSO4, and excess NiSO4 was washed with 2 volumes of water.
Finally the column was washed 2 volumes of Regeneration Buffer, and equilibrate
with 2 volumes of 1X PBS.
Protein in the samples was estimated by Bradford method using BSA as standard. A
standard curve of serially diluted BSA was prepared. Protein samples (2, 4, 6, 8 and 10
µl) were taken in clean test tubes. The volume was made 0.8 ml with water in all test
tubes. 0.8 ml of water in one test tube served as blank. 0.2 ml of Bradford reagent was
added to each test tube mixed well and incubated in dark for 5 minutes. The
absorbance was recorded at 595 nm. Total amount of protein in each sample was
calculated from the standard curve of BSA.
Western blotting was done to confirm the nature of over expressed proteins by using
monoclonal or polyclonal antibody against the over expressed protein. After running
the SDS-PAGE with pre-stained protein marker, the gel was transferred in the chilled
equilibration buffer. The PVDF membrane was cut and activated by immersing it in
chilled methanol for 10 min. In western blotting cassette, all the components were
51
placed in the following order from the cathode to anode end; sponge, whatmann filter
paper, gel, activated membrane, whatmann filter paper and sponge.
In wet transfer method the cassette was filled with transfer buffer and the transfer was
done for 20 hr at 20 m.v. After 20 hr the membrane was transferred to 3% BSA
solution and incubated overnight at 4°C or for 1 hr at room temperature with constant
rocking. Blocked membrane was further washed for three times for 10 min each with
PBS-T solution and incubated with diluted primary antibody (1:1000) for 1 hr at room
temperature with constant rocking. Washing was followed by three times for 10 min
each with 1X PBS-T. Washed membrane was incubated with diluted secondary
antibody conjugated with HRP (1:1500) for 1 hr at room temperature with shaking.
Detection was done by adding the diluted DAB substrate and hydrogen peroxide.
2.4.9 Electroelution
Electroelution of protein from the SDS-PAGE gel for further use was done by
Electroelution apparatus obtained from Bio-Rad. The SDS-PAGE gel was stained with
Coomassie blue staining for 30 min and destained for 1 hr. The purified band of
protein of interest was sliced from the gel and cut into small pieces. The gel pieces
were placed within the poly carbonate tubes and the apparatus was assembled. After
the final set up, all the air bubbles were removed from the tubes and the tubes were
placed within the gel tank. 1liter of electrodialysis buffer was poured in to the tank,
and electroelution was done at 20 volt, for 8-10 hr. Afterwards the destained gel pieces
were removed from the tube, and the eluted protein solution of blue colour was
recovered from the lower trap of the tube. For further use the eluted protein was
lyophilized.
2.4.10 ELISA
ELISA or Enzyme Linked Immuno Sorbent Assay was done to detect the level of
antibody titre raised in rabbit. Antigen or the purified protein was diluted to 0.1 µg/25
µl (4.0 µg/ml) in coating buffer (0.1 M NaHCO3, pH 8.6), and each well was coated
with 25 µl (0.1 µg). The plate was covered with plastic film and incubated at 4°C
overnight. On next day the coating solution was washed out of wells and the wells was
52
washed twice vigorously with distilled water. The wells was blocked by the addition
of 50 µl 3% BSA/PBS solution and followed by the incubation for 1 hr at room
temperature, wrapped in plastic or in a moist sealed container. The blocking solution
was shaked off (without washing or drying) and washed with 1X PBS for three times.
The primary antibody dilution in 1X PBS was done in 1:10, 1:100, 1:1000 1:10,000
ratio. Serially diluted primary antibody solution of 25 µl was added in each well, in
triplicate manner. The plate was wrapped in plastic wrapper and incubated at room
temperature for 1 hour. The primary antibody solution was shaked off and the wells
were washed with 1XPBS. 25 µl of 1:1000 times diluted secondary antibody
conjugated with HRP was added to each well and the plate was wrapped in plastic
wrapper. 1 hr incubation was done at room temperature, followed by three times
washing with 1X PBS. TMB substrate was diluted and added to each well, followed
by the addition of hydrogen peroxide and detection of signal at 495 nm.
53
2.4.12 Raising polyclonal antibody in rabbit.
Polyclonal antibody was raised in rabbit, following all standard animal ethics and
maintaining the rabbit in a hygenic condition. For raising antibody against one of the
expressed protein, a male white rabbit of 500 gm weight was obtained from the local
suppliers. Before raising the antibody, 1 ml of blood was collected from the central ear
artery of that rabbit to obtain the pre immune serum. The antigen/protein was purified
by affinity chromatography and eluted from the gel, followed by 5 hr lypholization.
Concentration of the the lypholized protein was checked. 500 µg of lypholized protein
was diluted to 1 ml with 1X PBS and mixed with 1ml of complete Freund’s adjuvant
(CFA). The mixture was vortex vigorously to obtain an emulsified solution, which was
injected intramuscular way to the rabbit. All the subsequent injections were done by
using Incomplete Freund’s Adjuvant (IFA). The following schedule (Table 2.2) was
maintained to obtain maximum antibody titer.
All the blood except the final bleed was collected from the central ear artery with a 19
gauge needle. The final bleed was drawn from the heart, and allowed to clot and
retract at 37°C overnight. The clotted blood is then refrigerated for 24 hr and the
serum clarified by centrifugation at 2500 r.p.m for 20 min. Straw colored serum was
decanted and aliquted to store in -20°C.
54
2.5 Enzymatic methods
The chitinase activity against β-1-4 glycosidase bond was determined with soluble
artificial substrate 4-methylumbelliferyl-tri-N-acetylchitotrioside, which release
fluorosence product. The substrate was dissolved in pure DMSO and 1 mM stock was
prepared. In a typical reaction volume of 300 µl, 200 mM of sodium phosphate buffer
(pH 7.2) varying amount of substrate (1-10 µM) was added with required amount of
purified enzyme. The reaction was continued and the mixture was incubated for 15-30
min at 25°C. 100 µl of 200 mM sodium carbonate solution was used to stop the
reaction. DMSO was used as negative control.
Inhibitor studies were done against the chitinase enzymes by using three previously
reported methylxanthine/methylxanthine derivative compounds namely Pentoxifylline,
Theophylline and Caffeine. 10 mM Stocks were prepared by dissolving all the
55
inhibitors in double distilled water. 300 µl of reaction mixture was prepared with 1
mM 4-methylumbelliferyl-tri-N-acetylchitotrioside (1-5 µM), 5 µg of purified enzyme
and 200 mM sodium phosphate buffer (pH-7.2) containing different amount of
inhibitors (50-150 µM). Reactions were stopped after 15-30 min of incubation at 25°C
by adding 100 µl of 200 mM sodium carbonate solution. Product formation was
determined by measuring fluorescence emission (excitation at 360 nm and emission at
450 nm) using Simple reading mode of Varian spectrofluorometer. All the results were
analyzed using single inhibitor-single substrate mode of inhibitor analysis programme
of Enzyme Kinetics module of Sigma Plot software. Ki values of the used inhibitors
and mode of inhibition was calculated using Lineweaver-Burk plots.
Colloidal chitin was prepared as described in materials. Colloidal chitin and chitin dust
was used as insoluble natural substrate to understand the substrate cleavage pattern of
chitinases. 100 µl of colloidal chitin or 1 mg of chitin dust was washed with and
resuspended in 200 mM sodium phosphate buffer (pH-7.2). Required amount of
purified enzymes were added to the mixture and the reaction mixture was incubated
for different time span (1 hr - 24 hr) at room temperature with constant agitation. 200
mM sodium carbonate solution was added to stop the reaction. The mixture was
56
centrifuged at 10,000 r.p.m. for 10 min and the supernatant was analyzed by reverse
phase HPLC. As mobile phase water-acetonitrile was used, along with C18 column as
the stationary phase, and the elution was done by distilled water with a flow rate of 1
ml/min. Product detection was done at 210 nm at room temperature. Standard
oligosaccharides were used to standardize the elution peak profile.
Entamoeba histolytica (NIH strain) and Entamoeba invadens (IP1 strain) were
maintained axenically in TYIS-33 medium at 37°C, and 25°C respectively.
Entamoeba histolytica cells were routinely sub cultured in every 48 hr, depending on
the cell confluency. Entamoeba invadens cells were sub cultured twice in a week.
For the initiation of encystation log phase Entamoeba invadens trophozoites were
harvested and centrifuged at 1500 r.p.m. for 3 min to pellet down. Single washing with
TYIS-33 medium was followed by three washes with encystation medium. Cells were
counted by hemocytometer, and diluted to reach the final concentration of 107 cells per
ml. Cells were resuspended in Nunc flask filled with 40 ml of encystation medium and
incubated at 25°C. Clumping of encysting cells was observed after 12 hr, and mature
cyst was obtained within 60-72 hr. Maturity of the cyst was determined by CFW
staining. For different time dependent studies, encysting cell of different hr was
collected and treated as per the requirement.
Entamoeba invadens mature cysts were obtained from 72 hr encystation culture, and
washed with encystation medium. This population contains encysting trophozoites as
well as mature cyst. Washing with chilled 1X PBS was followed by 5 min incubation
with 0.05% SLS solution. Only the mature cysts will remain in the solution, while the
trophozoites or encysting cells were get lysed by SLS. Viability of the mature cyst was
57
verified by Trypan Blue exclusion. The mature cysts were washed several times with
1X PBS to wash away the SLS. Finally the pellet was washed twice with TYIS-33
medium and resuspended in it. Within 12 hr of incubation emerging trophozoites were
observed under phase contrast microscope.
58
Olympus Flowview FV1000 confocal Laser scanning microscope with argon and
HeNe lasers was used for sample analysis, using respective laser and fluorescence
channels.
A cell freezer was pre-chilled with isopropanol at -80°C for 24 hrs before starting the
cryopreservation procedure. Fresh log phase axenic culture of Entamoeba was chilled
on ice for 5 min. Chilled cells were centrifuged at 1500 r.p.m. for 3 min, and the
medium was decanted. The cells were resuspended in 2 ml of solution I in a 2 ml tube.
From there cell suspension of 0.5 ml was transferred in 1 ml cryotubes. Prepared
solution II (15% Cell culture grade DMSO solution) of 0.5 ml was then added to that
cryotubes to make up the final volume to 1 ml. Filled tubes were incubated in 37°C
incubator for 15 min, followed by rapid cooling. The cryotubes were directly
transformed into the cell freezer, kept at -80°C. The cell freezer (with the cryotubes)
was kept in -80°C for 48 hr, followed by a liquid N2 storage.
Cryo-preserved tubes were collected from the liquid N2 tank, and immersed in 37°C
water bath immediately to thaw the medium. The tubes were incubated for 10 min in
37°C, and used to inoculate 15 ml culture tube filled with pre-warmed TYIS-33
medium. The culture tubes were kept in 37°C incubator in slanted position for 48 hr
without any disturbance. After 48 hr, fresh medium was added and again the tubes
were incubated for another 24 hr more, after which subculture was started.
59
60
Chapter 3
3.2 Introduction
Entamoeba histolytica has a haploid genome, though its ploidy was not maintained well
throughout the life cycle. In a previous observation E. histolytica DNA is being found to
resolved in 6 to 9 bands between 300 and 2000 kilobases, distributed in 16-22 large and
up to 31 small chromosomes (Valdes et al., 1990). The total genome of E. histolytica
HM1: ISS strain is find to be more than 20 Mb containing 14 chromosomes while most of
its extra-chromosomal DNA content was made up of rDNA. Amoebic genes are
reportedly have limited numbers of introns with short and intergenic sequences. The
completed E. histolytica genome project was a collaborative effort involving both TIGR
and the Sanger Institute Pathogen Sequencing Unit. The random shotgun strategy was
used for E. histolytica genome sequencing. Randomly 2.0 kb average insert size plasmid
was prepared to make 6-8 kb pHOS2 libraries of the E. histolytica genome as well as a
larger-insert BAC library. Sufficiently large numbers of randomly selected clones
obtained from sequencing libraries and sequenced from both ends to generate a high
quality draft (Loftus et al., 2005).
Several phylogenomic analyses support the proposal of lateral gene transfer among the
Entamoeba genome, specifically among the metabolic repertoire. The genome encodes a
large number of novel receptor kinases and contains expansions of a variety of gene
families, including those associated with virulence. E. invadens a reptilian parasite
reportedly mimic the symptoms of human enteric pathogen E. histolytica, and used
63
widely as a model of encystation study because E. histolytica is not amenable to encyst in
axenic culture. In pulse field gradient electrophoresis of E. invadens, 4 to 5 bands
between 300 and over 2000 kb were resolved and discriminated in 4 large and 6 small
chromosomes (Valdes et al., 1990). At the nucleotide level E. invadens genome has on
average 60% sequence identity with that of Entamoeba histolytica with a 70% genic
identity and 50% intergenic identity. Highly repetitive sequences such as of rRNAs,
tRNAs, CXXC-rich proteins, and Leu-rich repeat proteins are found to be in the E.
invadens genome as they are in E. histolytica genome. E. invadens proteins involved in
amoebic virulence, vesicular trafficking, signal transduction, and drug resistance are
found to have homologs in E. histolytica genome (Wang et al., 2003).
Presence of a single chitinase gene in E. histolytica genome has already been reported,
followed by its sequence analysis (de la Vega et al., 1992). Presence of multiple
chitinases within E. invadens genome was also reported (Wang et al., 2003) and was
partially characterized (Villagomez Castro and Lopez-Romero, 1996). Occurrence of
multiple chitinases in E. invadens genome is not an isolated incident, as gene multiplicity
is common among kinetoplastid protozoan’s (Landfear et al., 1983) expressing same
protein (Seebeck et al., 1983). Multiplicity of other chitin-binding domain may occur due
to domain shuffling as it happens in other fungal and bacterial chitinase and carbohydrate
binding proteins (Shen and Jacobs-Lorena, 1998).
3.3 Results
Chitinase (E.C 3.2.1.14) is a well-documented enzyme and close to lysozyme group, that
breaks down the β (1-4) glycosidic bonds of polysaccharide chitin. The enzyme belongs
to glycosyl hydrolase group, and was classified into two well distinct families depending
on its three dimensional structure. Family 18 includes the chitinases and homologous
chitin binding proteins termed as chito-lectins from viruses, bacteria, fungi and animals as
well as classes III and V from plants. Family 18 enzymes were known to adopt the TIM
(triosephosphate isomerase) barrel structure consisting of strongly conserved (α/β)8 motifs
containing separate chitin-binding domains in the carboxyl terminal region. Chitinases
belonging to this family shows a well-conserved catalytic domain sequence containing
several key aromatic amino acids needed for substrate binding. Family 19 chitinases are
64
identified mostly from plants (classes I, II and IV), nematodes, and some bacteria
(Funkhouser et al., 2007).
Eh chitinase sequence (Accession no. XP652205) obtained from the already published
genome, and used to perform BLAST within the E. invadens genome. Three nucleotide
sequences, named as EiChit1 (Accession no. AAB52724), EiChit2 (Accession no.
ABC59330) and EiChit3 (Accession no. ABC59331) were obtained from E. invadens
genome. ORF finder was used to detect the longest reading frames, and respective protein
sequences were selected for further studies. Protein sequences of other chitin binding
proteins of E. invadens such as Jacob1 (Accession no. AF175527) and Jessie3b
(Accession no.ABC59329) were obtained from the NCBI database. All accession no. are
enlisted in appendix A.
From the obtained nucleotide and protein sequences, we gathered several preliminary
informations using ExPASy proteomics server and its primary structure analysis tool.
Theoritical mol wt, pI, and other primary sequence related measurements of each protein
are presented in Table 3.1. Secondary structures containing α helices, β sheet and random
coils were obtained from Predictprotein server, and their probable percentages are
tabulated in Table 3.1.
65
Hydropathy plot or hydrophobicity of the proteins were calculated using Kyte-Doolittle
scale (Kyte and Doolittle 1982). SignalP, TMHMM, TMpred, nnPredict servers were
used to understand the localization and vesicular transportation of these chitinases due to
posttranslational modifications. All the Entamoeba chitinases found to belong to the
family 18 glycosyl hydrolase, by Conserved domain search programme.
Family 18 of glycosyl hydrolase have many members from bacteria, insect, and higher
animals including humans, and bear some signature domain and conserved motif within
the catalytic domain. Protein sequence of Ei Chitinase 1 was used as query sequence in
blastp programme to search similar sequences within the non-redundant database. Twenty
sequences were selected depending on the highest score and lowest e value. Accession
number of retrieved sequences are listed in appendix B and Fragments of aligned
sequences are represented in Fig.3.1.
66
Fig. 3.1: Mutiple sequence alignment of 20 chitinases using ClustalW. Fragments of catalytic
domain sequences presented here and four- consensus motif I, II, III and IV indicated within box.
All default gap value and penalties (such as Open gap is -12, extend gap is -2, end gap -1,
and gap distance is 4) were used for alignment along with the default Gonnet matrix. The
aligned sequences show four consensus motif containing consensus aromatic residues
such as VGGW (domain I), DWEYP (domain II), MTYDF (domain III), and VGM
(domain IV).
Twenty chitinase sequences used to prepare the phylogenetic tree by minimum evolution
method with bootstrap values using MEGA software (Kumar et al., 2007; Rzhetsky and
Nei 1992). The bootstrap consensus tree inferred from 1000 replicates is taken to
represent the evolutionary history of the taxa analyzed (Fig 3.2) (Felsenstein, 1985).
The tree was drawn to scale, with branch lengths as the evolutionary distances computed
using the Poisson correction method (Zuckerkandl and Pauling, 1965), and is in the units
of the number of amino acid substitutions per site. All positions containing gaps and
missing data were eliminated from the dataset (Complete deletion option). Phylogenetic
study in relation with other well-known chitinases clearly predict the difference between
67
the sequence of bacteria (IV), protozoan (III), insect (II) and higher animals (I), clustering
them in different clades.
Fig. 3.2: The Phylogenetic tree presented constructed using Mega (version 4) software. Minimum
evolution method was used with Poisson correction for multiple amino acid substitution and with
1000 random bootstrap replicates. The Fig. shows the minimum evolution tree. Alignment of
amino acid sequences of 20 different chitinase was done using ClustalW. The chitinases comprise
four clusters denoted as I, II, III and IV (discussed in the text). The scale at the bottom left is in
units of amino acid substitutions per site. Bootstrap values ≥ 50% are shown.
For RT-PCR, different primers were designed by using Oligo Perfect server (Invitrogen).
All the nucleotide sequences were uploaded, and primers were designed to amplify ~400
bp long fragment of the C terminal of the protein of the Ei Chitinase 1, 2 and 3 (Table
3.2). For cloning and expression in bacterial system, primers were designed using Oligo
perfect software (Invitrogen) excluding the predicted N-terminal signal sequences for
both Eh and Ei Chitinases (Table 3.3). For expression in bacteria, Methionine codon
(ATG) was added to the 5’ of the every forward primer.
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Table 3.2: Primer sequence (all 5’Æ3’) used for RT-PCR.
Table 3.3: Primer sequences (all 5’Æ3’) used for expression study. Incorporated restriction sites
were underlined and italicized.
E.invadens Chi1CBD-R-Cat Sense GGATCC ATG TGT GAA GGA CTC GAC AAC GG
E.invadens Chi1Cat Sense GGA TCC ATG AAG GTT GTC TCG TAT TAC ACC
Antisense CTC GAG TTA GCA ACC GAT CAA GCT CTT TC
E.invadens Chi2Cat Sense GGA TCC ATG AAG GTG GTT GGG TAC TAT AC
E.invadens Chi3Cat Sense GGA TCC ATG AAA GTG ATT GGA TAC
Antisense CTC GAG TTA GTT TTT CAA CTC ATC GAT C
E.histolytica Chi1CBD-R-Cat Sense GGA TCC ATG CAC AAC TGT GAA GGT C
Antisense GCG CTC GAG TTA ACA TTT CTC AAT TAG
E.histolytica Chi1Cat Sense GGA TCC ATG AAA ACA GTT GCT TAT TAT AC
Antisense GCG CTC GAG TTA ACA TTT CTC AAT TAG
E. invadens JacobCBD Sense GGA TCC ATG CAA ACA TGT ACA ATT GAT GG
Antisense AGA TCT GTA GTT TCT TCT TTG TAA CCA CAT G
E. invadens JessieCBD Sense GGA TCC A TGC AAA CAT GTA ACG GGC T
Antisense AGA TCT GTC CAC AAA ATA GTC TCC TGG TT
E.invadens Chi1R-Cat Sense AGA TCT AAG TCG TCT GAG GAA CCA CTC
Antisense GTC GAC TTA GCA CTG ATC AAG CTC TTT TTC
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3.3.5 Homology modelling
A B
Fig. 3.3: Predicted models of EiChit1 (yellow) and EChit2 (green) [A], and EiChit2 (green) and
EiChit3 (yellow) [B] were presented in ribbon style, superimposed using Chimera. Top views of
the TIM barrel structure of the superimposed models are shown. It is evident that the predicted
models of all three chitinases are very similar except few loop regions.
70
Due to the conserved three-dimension structure, it was assumed that all E. invadens
contain similar primary sequences throughout the catalytic domain. To identify the
extension of sequence similarity within different species of Entamoeba, sequence
alignment was done (Fig 3.5).
Fig. 3.5: Aligned catalytic domains of Entamoeba chitinases. Entamoeba histolytica (XP652205),
Entamoeba invadens (Chi 1 -AAB52724, Chi2- ABC59330, and Chi3- ABC59331) and
Entamoeba dispar (XP001735375) chitinases were aligned using ClustalW server of EMBL.
Conserved amino acid residues of active site of the enzyme were indicated within box. Identical,
similar and somewhat similar amino acids are indicated with asterics (*), colon (:) and dot (.).
71
NAG molecule were taken from hevamine-chitrotetrose complex and the +2 subsite
binding NAG molecule was taken from S. marcescens chitinase A-chitotriose complex.
Docked molecule was visualised after the energy minimization with Chimera (Pettersen
et al., 2004) (Fig.3.6). Interaction of the sugar residues with the conserved aromatic
amino acid residues was observed. The bound complexes were scored using SCORE
(Wang et al., 1998) and they gave pKd values of 5.38, 4.58, 5.27. Hence it could be
clearly seen that the protein prefers occupancy at the +1, +2 subsite rather than at the -1
subsite alone, resulting in the cleavage of dimers from the substrate end.
Fig. 3.6: Molecular docking of NAG5 within the catalytic cleft of predicted model of E. invadens
Chitinase 2. Penta N-acetyl Glucosamine (NAG5) bound to the modeled catalytic cleft of
E.invadens chitinase2. The thin line structure in cyan represents the oligo chitin (NAG5) with the
number beside each sugar ring representing the subsite. Amino acid residues of the active site
W84, N85, D128, E130, Y131, Y203, D204, R253 and E282 interacting with the sugar molecule
are indicated. The surface is shown to highlight the catalytic cleft. The image is generated using
PyMOL (DeLano Scientific). Sugars are numbered -3 through +2 from the non-reducing end as is
the current convention.
3.4 Discussion
Total genome sequencing of E. histolytica reveal the presence of single chitinase, while
three chitinases were identified in its reptilian counterpart E. invadens. To understand the
enzymatic profile of these enzymes, molecular characterization is a necessity, which is
often proceeds with the in silico characterization. In silico characterization includes the
72
understanding of phylogenetic relation, reorganization of structural aspect, and its
behaviour with the probable substrate.
Different Bioinformatics software were used to identify the preliminary aspects of these
proteins, such as the theoretical mol. wt. and pI calculation. Different range of pI signify
the overall surface charge and may involved with substrate binding efficiency, as lower pI
value increase the chitin binding capacity.
All four chitinases show the presence of N-terminal signal sequences with a chance of
post-translational cleavage of approx 15 amino acids, which signify the chance of protein
transport and occurrence of specific destination. Maximum chance of cleavage was
calculated mostly between polar-polar and nonpolar-nonpolar residues such as Serine-
Glutamine (EiChit1), Glycine-Leucine (EiChit2), Glutamine-Lysine (EiChit3) and
Alanine-Histidine (EhChit1). nnPredict server predicted the secondary structures which
shows more or less similar distribution of helices, sheets and random coils within
EiChit1, 2 and EhChit1, which is different than EiChit3. This difference may change the
three dimensional orientation, resulting in different substrate binding efficiency.
Phylogenetic analyses have shown that all known family 18 chitinases contains four
conserved regions within the catalytic domain (de la Vega et al., 1998). Conserved region
I is KXXXXXGGW, where X is a non-specific amino acid. Conserved region II is
comprised of FDGXDLDWEYP, which is believed to be located in the catalytic region of
the enzyme and the residue E is a putative proton donor in the substrate hydrolysis.
Conserved regions III and IV are MXYDXXG and GXXXWXXDXD, respectively.
Aligned sequences shows consensus motif with polar and aromatic residues such as
VGGW (domain I), DDWEYP (domain II), MTYDF (domain III) and VGM (domain
IV). All Entamoeba chitinases along with the representatives of other group confirms the
presence of such conserved motifs, which further strengthen their position in family 18 of
glycosyl hydrolase.
Phylogenetic investigation was done using the MEGA software and minimum evolution
method. Bootstrap consensus tree shows four different clusters named as I, II, III and IV
containing vertebrate, insect, protozoa and bacteria chitinase respectively. All Entamoeba
chitinases clustered among a distinct clade separated from other group ruling out the
possibility of lateral gene transfer, which happened to be the reason behind the origin of
most Entamoeba gene (Samuelson, 2002). The distribution of Entamoeba chitinases
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clearly show that they are not evolutionary related to any bacterial, fungal, or insect
chitinases and have no bacterial ancestors. Among the different clades, Entamoeba
chitinases show comparatively high resemblance with the chitinases of evolutionary
advanced organism such as with insects and vertebrates. This similarity explained by
convergent evolution of different proteins under same evolutionary selective pressure. All
three E. invadens chitinases shows different time point of origin and remain separated
from each other. E. histolytica and E. dispar probably originated at in same evolutionary
period. Among three E. invadens chitinases, Ei Chitinase 3 probably evolved primarily,
followed by Ei Chitinase 2 and Ei Chitinase 1. Addition of extra domain may occur in
evolutionary pathway, to serve some other purpose such as substrate binding, rather than
only substrate hydrolysis. The origin of multiple chitinase in E. invadens probably occur
long ago to serve different purposes during encystation and excystation, while in
advanced E. histolytica and E. dispar, a single chitinase probably found to be enough to
serve all requirements, explain the absence of other two chitinases.
Homology modelling of E. invadens chitinase was done to identify the active residues
responsible for substrate hydrolysis. Human chitotriosidase used as template for
modelling study due to the high sequence similarity and score value. All three Ei
chitinase models were superimposed with each other without any significant structural
distortion. In spite of different secondary structure distribution, their tertiary conformation
of the catalytic domain remains same except of some loop regions. Multiple alignment of
only the catalytic domain of Entamoeba chitinases detects identical aromatic residues,
which may play important role in binding with negatively charged substrates.
Molecular docking with oligomers molecular was done to characterize the role of those
aromatic residues. Docking was done by superimposition using penta and hexa NAG
molecule. Docked protein shows preference for +2 and +1 subsites along with -1 subsite.
Tyr131 and Glu130 along with Tyr203 and Asp204 may play some important role in
substrate binding.
The hydrolysis reaction mechanism may follow the common anchimeric stabilization
hydrolysis mechanism of family 18 chitinases where the substrate is distorted to a boat
conformation by forming the oxazolinium intermediate. Chitinases exhibiting this mode
of substrate hydrolysis, can be inhibited by allosamidine, the pseudosaccharide molecule
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which mimic the oxazolinium group. Other inhibitor molecules mimicking the basic
structure of allosamidine also found to inhibit such enzymes to some extent.
3.5 Conclusions
• Nucleotide sequences of all the Entamoeba chitinases were obtained from the
complete or ongoing genome project.
• All the Entamoeba chitinases belongs to the family 18 chitinases and contain
TIM barrel structured catalytic domain.
• All Ei and Eh chitinases contain signal sequences and destined to be in the
outside of the cell membrane or in vesicle.
• Primers for cloning and RT-PCR were designed using bioinformatics tool.
• Phylogenetic studies shown high similarity between all Entamoeba chitinases,
while they do not show enough resemblance with other bacterial, fungal or
plant chitinases eliminating the chance of lateral gene transfer. Similarity with
insect or vertebrate chitinase may occur due to convergent evolution.
• Homology modelling of all the Ei chitinases were done, which show highly
similar catalytic cleft structure, with few exception at loop regions.
• Molecular docking of penta chitosaccharide (NAG5) within Ei Chitinase 2
model shows the substrate binding sites and possible substrate cleavage
pattern.
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76
Chapter 4
Molecular cloning and expression of all three Ei and single Eh chitinases are done in
bacterial system and discussed in this chapter. All four genes were fished out by PCR
amplification and subjected to further cloning. For single step cloning, pGEM-T
vector was used followed by the sub cloning in pQE30 vector for bacterial expression.
Recombinant proteins were expressed with N-terminal His tag, after induction with
IPTG. Over-expressed Protein of 56 kDa (EiChit1 and EhChit1) and 45 kDa (EiChit2
and EiChit3) were confirmed with monoclonal anti His antibody and purified to 80-
90% homogeneity with Ni-NTA agarose beads
4.2 Introduction
Entamoeba histolytica, the amoebic parasite encyst and excyst to survive through the
harsh environment and propagate within the host with ease. This transformation
requires the synchronized initiation of several pathways, which finally completed the
chromosomal rearrangement, compartmentalization of several stage specific proteins,
creation of the chitinious cyst wall. Detail understanding of these procedures is still in
its infancy. In encysting Entamoeba invadens, several proteins were found to be
controlled transcriptionally (Coppi and Echinager, 1999; Ehrenkaufer et al., 2007) and
thus predicted to play an important role. Among these transcriptionally regulated
proteins, very few were characterized in details. Molecular characterization of these
proteins may enlighten their specific roles and may initiate the search of new drug
molecules against them (Laughlin et al., 2005). Protozoan chitinase which was found
to play important role in parasite transmission or host invasion, used as a potential
drug target, and its inhibition in these organism were found to produce less virulence
or low transmission ability within host (Shahabuddin et al., 1993; Welburn et al., 1993
and Rogers et al., 2008).
Chitinase belongs to such transcriptionally regulated protein group (de la Vega et al.,
1997) and required during encystation, as the well known chitinase inhibitor
“Allosamidine” was found to inhibit the E. invadens encystation (Villagomez Castro et
al., 1992). Though its role during excystation was not clear, degradation of chitinious
cyst wall was proposed to be the aim of these enzymes. Genome wide study of E.
histolytica reveals the presence of single chitinase, (Loftus et al., 2005) while E.
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invadens was reported to have a complete chitinolytic system consisting of three
chitinases (Wang et al., 2003). Cloning and expression of three Ei chitinase and one
Eh chitinase was done in bacterial system to characterize these enzymes.
4.3 Results
Fig. 4.1: Schematic diagrams of E. histolytica (EhChit1) and E. invadens (EiChit1, EiChit2
and EiChit3) chitinases used for cloning and expression. CBD represents N-terminal chitin
binding domain of approximately 60 amino acids. R represents heptapeptide serine rich linker
connecting CBD and catalytic domain. Catalytic domain displays the longest C-terminal
domain responsible for substrate hydrolysis.
PCR amplification was done using the Expand High Fidelity PCR system. Ei chitinase
1, 2, 3, and Eh chitinase 1 were amplified by using the following cycle, of 95°C for 5
min, 35 cycles at 92°C for 30 s, 55°C for 30s, 68°C for 1.5min (1min for 1000bp ) and
then by a final extension at 72°C for 5min. All the amplified products of
approximately 1500 bp (EiChit1 and EhChit1) and 1200 bp (EiChit2 and EiChit3)
were checked in 0.8% agarose gel with 1kb DNA ladder (Fig. 4.2)
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Fig. 4.2: Amplified EiChit1, EiChit2, EiChit3 and EhChit1 and 1kb DNA
marker. Amplicons are shown with black arrows
The amplified products were purified further by gel elution and used to determine the
product concentration at 260nm. Approx 75 ng of PCR product (approx length 1.2-
1.5kb) was ligated to 50 ng of supplied vector mixture in 3:1ratio using the following
equation; where vector size is 3kb, and insert:vector ratio is 3:1.
The ligated products were transformed into competent E. coli, DH5α cells. Retardation
of plasmid DNA from white colonies in comparison to blue colonies in agarose gel
electrophoresis was used as a preliminary screening for recombinants, followed by
restriction digestion with BamHI and XhoI.
Digested plasmids were run in 0.8% gel in comparison to undigested plasmids, and
inserts of expected length were detected (Fig.4.3). Sequencing was done for
confirmation.
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Fig. 4.3: Agarose Gel electrophoresis of restriction enzyme digested TA clones of EiChit1,
EiChit2, EiChit3 and EhChit1. Lane1- TA-EiChit1, Lane 2- Digested TA--EiChit1, Lane 3-
TA-EiChit2, Lane 4- Digested TA-EiChit2, Lane 5-TA--EiChit3, Lane 6- Digested TA-
EiChit3, Lane 7- TA-EhChit1, Lane 8- Digested TA-EhChit1. Lane M represents 1kb DNA
Marker. Inserts are shown with black arrows.
Sub-cloning of all the chitinases was done in bacterial expression vector pQE30
(Qiagen, Germany) for expression study. BamHI/XhoI digested chitinase fragments
were cloned in the BamHI and SalI site of the pQE30, as SalI and XhoI are compatible
with each other. Digested vector and insert were gel purified and ligated in 3:1 ratio.
Chemically competent XL1 Blue cells were transformed with ligation mixture.
Recombinant colonies and confirmed by the restriction digestion with BamHI and
HindIII, as the SalI and XhoI sites get destroyed followed by the ligation. Digested
plasmids were run in 0.8% agarose gel, and cleaved bands of expected lengths were
identified by comparing to 1kb DNA ladder (NEB) (Fig. 4.4)
Fig. 4.4: Agarose gel electrophoresis of pQE30 clones of EiChit1, EiChit2, EiChit3 and
EhChit1, digested with BamHI and HindIII. Lane M represents 1kb DNA marker. Inserts are
shown with black arrows.
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4.3.4 Expression of Entamoeba chitinases in bacterial system.
Recombinant proteins were expressed in M15 (pREP4) strain of E. coli containing the
lac repressor expressing plasmids (pREP4). The pREP4 plasmid is derived from
pACYC and multiple copies of pREP4 were present in the host cells ensuring the high
levels of the lac repressor protein and tight regulation of recombinant protein
expression
Confirmed pQE30 clones were used for transforming chemically competent M15
cells, and plated on Amp-Kan LB plate. Single colonies were selected and used for
expression study. Freshly grown log phase culture was induced with 1 mM IPTG
solution, followed by 5 hr post induction incubation at 37°C. The total bacterial
proteins were electrophoresed in 12% SDS-PAGE gel in comparison with un-induced
total protein, to detect the induced band. The induced lanes showed the bands of
expressed proteins, at expected region. Induced bands of expressed proteins were
observed at different molecular weight which is slightly higher than their expected
length (Fig. 4.5) (EiChit1- 65 kDa, EiChit2- 45 kDa, EiChit3- 45 kDa and EhChit1- 65
kDa).
The over expressed protein contains 6 Histidine residue at their N-terminal. Further
confirmations of expressed proteins were done by western blot using monoclonal Anti
His antibody as primary antibody (Fig. 4.6). HRP conjugated Protein A was used as
secondary reagent, and Di-amino-benzoic acid (DAB) detection system was used for
83
band development. Single band of His tagged proteins were observed in total protein
fraction of induced M15-Entamoeba chitinase clones at their respective region.
Fig. 4.6: Western blots of expressed, EiChit1, EiChit2, EiChit3 and EhChit1. Total bacterial
proteins from induced clones were used, and over expressed bands were detected with
monoclonal anti His antibody and marked with black arrows.
The N terminal 6xHis affinity tag of pQE30 system was used for purification, with Ni-
NTA agarose beads by affinity chromatography. Low immunogenicity and small
length of 6xHis tag do not interfere with protein compartmentalization, folding and
activity. Presently Nitrilotriacetic acid (NTA) (Qiagen) a tetradentate chelating
adsorbent, used solely for IMAC allowing the purification of very low amount of
protein, which results in more than 90% homogeneity in single step purification.
Expression profiling was done to identify the nature of expressed proteins. Induction
was done with 1 mM IPTG, followed by 5 hr post induction incubation at 37°C. The
induced bacterial pellets were sonicated and centrifuged to separate the soluble or
membrane bound proteins, and analyzed in 12% SDS-PAGE. Induced proteins were
found mostly in membrane bound fractions in every case.
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Level of expression in soluble fraction was further confirmed by the western blotting
of the recombinant proteins, using monoclonal anti His Antibody. The optimized post
induction incubation period and temperature was tabulated in Table 4.1.
Purification was done using Ni-NTA agarose beads, as mentioned in “Methods and
Materials”. In every case 200-250 mM was found to elute more than 90% of the bound
protein. Purification quality was determined by 12% SDS-PAGE, followed by staining
and destaining (indicated by black arrow in Fig. 4.7). Purified proteins were further
concentrated using centricon with 30 kDa cut off membrane (Pall Sciences) and
quantified with Bradford reagent using BSA fraction five (1mg/ml) as standard. Total
protein yield were tabulated in table 4.1.
Fig. 4.7: Single step purification profiles of EiChit1, EiChit2, EiChit3 and EhChit1.
Purified bands are marked with black arrows. Lane M- Protein marker
Table 4.1: Optimum IPTG Conc., post induction incubation temperature for maximum protein
yield and obtained protein.
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4.4 Discussion
Chitin, the polymer of GlcNAc molecule ranked as the 2nd most abundant one, after
cellulose and involved in different structural aspects of lower and higher group of
organisms. In many fungi and bacteria, occurrence of multiple chitinase is a very
common phenomenon.
Several human parasites were reported to contain single or multiple chitinases for
transmission through vectors, or to continue their life cycle. So these chitinases were
evaluated as a possible drug target either as a target molecule or a source for
transmission blocking antigen (Langer and Vinetz, 2001). Presence of any chitin or
any similar polysaccharide was not discovered in human system, multiple human
chitinase or chitinase like protein was discovered in last few years (Renkema et al.,
1995). Though their exact role was not characterized yet, their involvement in
different disease and genetic deficiencies made them an interesting molecule.
Other than Plasmodium (Sahabuddin et al., 1995) and Leshmania chitinase (Rogers et
al., 2008), Entamoeba chitinases were treated as a potential drug target to combat with
the amoebiasis. E. histolytica contain a single chitinase as Entamoeba dispar and the
exact role of this parasitic enzyme in its life cycle is still unknown. Interestingly the
genome sequence of “in vitro encystation model” E. invadens, a reptilian parasite,
comprises of three chitinases. Partial characterization of those enzymes was followed
by their isolation and purification from encysting cells (Villagomez Castro and Lopez
Romero, 1996). All Entamoeba chitinases contain conserved sequence throughout the
catalytic domain and belongs to family 18 of chitinase.
From the genome sequence it was observed that Eh chitinase contains a N-terminal
substrate binding domain, a C-terminal catalytic domain and a heptapeptide linker in
between. In a different note the Ei chitinase complex contains three enzymes having
different composition. Ei chitinase 1, the longest protein of them, has similar structure
86
as Eh chitinase, though the other two enzymes show a different structure by having
only the catalytic domain.
For PCR amplification, gene specific primers were designed and four chitinases were
fished out from the genomic DNA. Amplicons of EiChit1 and EhChit1 were found to
be of 1500 bp, while EiChit2 and EiChit3 produce 1200 bp amplicons. During PCR, a
mixture of amplicons were produced either containing “A” in their ends, or not. TA
vectors were used for cloning purpose by using those “A” overhangs as a linker. The
TA-chitinase clones were confirmed by restriction enzyme digestion and sequencing.
For expression purpose bacterial system was used for high protein yield and easy
maintenance. pQE30 expression system was used which contain N-terminal 6xHis tag
for one step purification. This 6 Histidine tag was rarely found to disturb the protein
folding and modulate the three dimensional structure, so removal of this tag from
expressed protein is not a necessity. All cloned fragments were cleaved by using
restriction enzymes and ligated to linearized pQE30 vector.
Confirmed pQE30 clones were used for protein expression in M15 strain of E. coli.
M15 strain contains pREP4 plasmid which produces the lac repressor to help tight
transcription regulation. IPTG was used as inducer. Over-expressed proteins were
observed only in IPTG induced bacteria. All four expressed proteins were analysed in
SDS-PAGE, and they appear to be a little bit heavier than their calculated weight due
to different SDS binding and different level of denaturation. Addition of 6xHis tag and
restriction sites also put some extra amino acid. Confirmation of expression was done
by western blot with monoclonal anti-His antibody.
All expressed proteins were identified primarily in membrane bound fraction, which
obstruct the purification and further characterization. Appearance of over-expressed
proteins in membrane bound fraction may occur due to high translation rate and
improper folding. So, IPTG concentration was lowered to reduce the expression level.
Lower post induction incubation temperature was introduced to inhibit the improper
protein folding. Finally all the proteins were obtained in soluble fraction after using
0.2-0.5 mM IPTG and 15°C as post induction incubation temperature.
Purification of soluble protein was done in single step using Ni-NTA agarose beads.
Nitrilotriacetic acid (NTA), the chelating adsorbent, occupies four of the six ligand
87
binding sites in Ni+2 ion. Two free sites in the coordination sphere of nickel ion
interact with the Histidine tag. The histidine residues become protonated below pH 4-
5, and no longer remain in bound condition with the nickel ions. But lowering the pH
of elution buffer may create improper protein folding, so high concentration of
imidazole was used instead, as 6xHis tagged protein couldn’t compete with them for
binding sites on Ni-NTA beads. Maximum amount of protein was eluted with 250 mM
imidazole solution.
4.5 Conclusions
• All Ei and Eh chitinases were amplified from genomic DNA using gene
specific primers.
• Amplified fragments were cloned in pGEMT vector using T/A overhangs.
• Recombinant clones were selected and confirmed by sequencing.
• Sub-cloning was done in pQE30 vector and confirmed.
• Recombinant protein expression was confirmed with 12% SDS-PAGE and
western blotting.
• Induction conditions were standardized to obtain maximum soluble protein.
• Purification of soluble proteins was done using Ni-NTA affinity
chromatography.
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Chapter 5
Enzymatic ability and efficiency of all four recombinant Entamoeba chitinases are
described in this chapter. All four recombinant chitinases demonstrate β (1-4) bond
cleavage activity against the soluble substrate, while both EiChit2 and EiChit3 are
found to be more efficient than other two. All Ei chitinases are able to bind the
insoluble substrate though two of them (EiChit2 and EiChit2) lacks the chitin binding
domain. All Entamoeba chitinase shows the ability of hydroxylation of insoluble
substrate and release dimers from the polymer substrate end as exochitinase. All Ei
chitinases get inhibited by methylxanthine derivatives at micro molar level and
inhibited in different manner which signifies the difference of their substrate binding
mode.
5.2 Introduction
Chitinases (EC 3.2.1.14) are glycosyl hydrolases that degrade the chitin, an insoluble
linear β (1-4) linked polymer of N-acetylglucosamine (GlcNAc) by cleaving its β (1-
4) glycosidic bonds. During the last few decades, chitinases have been considered as a
potential “bio-tool” because of their wide range of applications in different industries.
Applications of chitinases included the preparation of protoplasts from fungi, as
protective agent against plant-pathogenic fungi, and in the processing of biologically
active oligosaccharides. Enzymatically produced chito-oligomers have been in lime
light of interest in recent years due to their broad applications in medical and
agricultural fields as hypocholesterolemic, anti-hypertensive, antioxidative agents
(Chen et al., 2003) as well as for antibacterial and antifungal activity.
91
uncertain, where its role may include chitinious food degradation to immune response
against fungal infections (Gooday, 1986).
In general, chitinolytic enzymes can be divided into three categories, depending on its
substrate cleaving pattern. They are categorized as exochitinase demonstrating
cleavage activity only for the end of the chitin chain and endochitinase hydrolyzing
internal β (1-4) glycosidic bonds. Another class β-N-acetylglucosaminidase cleaves
GlcNAc units sequentially from the non-reducing end of the substrate. Naturally the
enzyme cleaves the substrate in random fashion, at internal locations over the entire
length of the polymer, producing soluble, low molecular mass multimers of GlcNAc,
such as chitotetraose, chitotriose, and chitobiose, with the smallest oligosaccharides
being predominant.
All members of family 18 chitinases shows structurally similar catalytic domain, made
up of 8 folds of consecutive α and β domains. Conserved aromatic amino acid residues
along with several polar residues are present throughout the catalytic cleft to assist the
substrate binding. Polymeric chain of chitin binds with the catalytic domain, but at
once only 4-5 monomer units are accommodated within the catalytic cleft of family 18
chitinases. As per the substrate subsite nomenclature, +1 and +2 subsites are found to
be important during enzyme-substrate interaction (van Aalten et al., 2001).
Most of the well studied chitinases belongs to glycosidase family 18 and found to
degrade the chitin with retention of the stereochemistry at the anomeric carbon (Tews
et al., 1997) (Honda et al., 2000). The classical reaction mechanism of substrate
molecule retention was observed in lysozyme (Davies et al., 1998) and amylase
(Uitdehaag et al., 1999). In chitinase this reaction is presumed to be initiated by
distortion of the -1 sugar ring and protonation of the glycosidic oxygen by a
protonated acidic residue. The classic lysozyme mechanism differs from chitinases,
during the subsequent nucleophilic attack involving the N-acetyl group of the -1 sugar,
rather than a carboxylate side chain on the protein (Terwisscha et al., 1995; Kobayashi
et al., 1996). The first step of hydrolysis results in cleavage of the sugar chain and
formation of an oxazolinium ion intermediate, which get hydrolyzed in next step to
complete the reaction (Brameld et al., 1998). The intermediate oxazolinium group
helps in electron transfer and balancing the charge distribution, during the hydrolysis.
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As, chitinases are important in fungi and insect for maintaining their life cycle,
inhibition of chitinases was found to be a good target for pesticide design. Several
reported inhibitors of family 18 chitinase, mimic the oxazolinium intermediate
structure. Allosamidine, is one such inhibitor, isolated from Streptomyces sp
reportedly inhibits family 18 chitinases at nanomolar level (Sakuda et al., 1986). This
pseudotrisaccharide is commercially unavailable, which leads the search for potent
and synthetic inhibitors. Arfidin, Argadin and Cl4 are few such synthesized inhibitors
reported in recent past, though the high molecular weight, very low cLogP value
(logarithm of its partition coefficient between n-octanol and water log) and presence
of several stereocenters made them poor lead as drug (Rao et al., 2005). A
comparatively new avenue was discovered to identify potential drug molecule by
screening drug library. In one such study, few molecules belonging to methylxanthine
group of chemicals shows some interesting inhibitory effect (Rao et al., 2005). These
molecules have several advantage over others, and can act as a lead for more
refinement, which may increase their efficiency as inhibitors.
5.3 Results
The natural substrate for chitinase is chitin or chitosan or the chito-oligomers. But due
to their crystalline structure and insolubility, they are not suitable to study the β (1-4)
bond cleavage efficiency of hydrolytic enzymes in vitro. To circumvent this problem
several artificial and soluble substrates were designed and made commercially
available for this purpose. These substrates contain small chito-oligomers, conjugated
with fluorescence or coloured compounds, which get cleaved and can be detected by
simple fluorogenic or colorimetric assay respectively.
93
Fig. 5.1: Chemical composition of 4-methylumbelliferyl-tri-N-acetylchitotrioside
The substrate was dissolved in pure DMSO to prepare 1 mM stock solution. Purified
proteins of required amount were added to a typical reaction mixture of 300 µl
containing 200 mM sodium phosphate buffer (pH-7.2) and 1-5 µM substrate. After 15-
30 min of incubation at 25˚C, the reaction was stopped by 200 mM of sodium
carbonate buffer. The product was quantified by Varian spectro-fluorometer, model
Cary-Eclipse equipped with a Xenon lamp. The concentration of the fluorescent 4-
methylumbelliferone product generated from these substrates was determined using
4MU as standards. One unit of chitinase activity was defined as the amount of enzyme
required to release 1 nmol of 4-methylumbelliferone (4MU) per second at optimum
temperature.
To identify the optimum pH for each Ei chitinases 200 mM sodium phosphate buffer
was used for pH range of 5-8 and 200 mM sodium acetate buffer was used for pH
range of 3-5. Optimum temperature was determined by repeating the assay within the
range of 4-45˚C.
EiChi2 and EiChit3 show comparatively high substrate affinity and high bond cleavage
efficiency than that of both EiChi1 and EhChi1. Optimum pH of all four chitinases was
94
found to be same for this artificial substrate. The optimum temperature differs among
EiChi2, EiChit3 and EiChit1.
To understand the substrate binding efficiency and the mode of ligand and enzyme
interaction, three insoluble substrates of different conformations were used in this
study. Spherical chitin beads and chitin dust were obtained commercially, while
amorphous colloidal chitin was prepared from chitin flakes (Hackman, 1962;
Jeuniaux, 1966).
Among the three substrates, spherical chitin beads has maximum exposed surface,
while the acid treated colloidal chitin have loosely bound fibrils (Fig. 5.2). Purified Ei
Chitinases were incubated with the substrates for 2 hr with constant agitation. The
bound and unbound fraction of proteins were separated by centrifugation, and
analyzed in 12% SDS PAGE (Fig. 5.3A).
95
Fig. 5.2: Images of Chitin beads, colloidal chitin and chitin dust, 10 x magnifications in phase
contrast microscope.
To identify the nonspecific binding, BSA (fraction five) was used as control. The
protein bands were scanned by densitometric scanner to determine the percentage of
bound proteins and the values were plotted after deduction of blanks (Fig. 5.3 B).
All three chitinases show different level of binding efficiency, while EiChit1
demonstrate maximum binding efficiency with all three different substrates. EiChit2
and EiChit3 were found to bind efficiently with chitin beads and other insoluble
substrates though they lack of the specific chitin binding domain. From the
densitometric scan, it can be concluded that the chitinases bind maximum to chitin
beads, and least to chitin dust.
Fig. 5.3: SDS-PAGE analysis of different substrate binding efficiency of three Ei chitinases
[A]. Graphical representation of percentage of bound protein calculated from band intensity of
bound fraction obtained from Densitometry scan [B].
96
of binding buffer of different pH. EiChit2 and EiChit3 bind most at pH 5, while
EiChit1 binds most at pH 4.
All recombinant Entamoeba chitinases show substrate binding affinity, which may
indicates their ability to degrade insoluble chitin. Colloidal chitin was used as
substrate as it contains more loose ends and comprises a relatively less compact
conformation. Fixed amount of colloidal chitin was incubated with fixed amount of
purified chitinases for 2 hr, in presence of 200 mM sodium phosphate buffer (pH-7.2).
The supernatant was collected by high speed centrifugation and analyzed in reverse
phase HPLC with C18 column and water-acetonitrile mobile phase. Elution was done
with water and product detection was done at 210 nm. Standard chito-oligomers were
used to identify the product structure. Product profile of colloid chitin was plotted
using multi-curve plotting option of Origin 8 software (Fig 5.4). All three chitinases
degrade colloid chitin and predominantly produce chitobiose, which classify them as
exochitinase. EiChit2 and EiChit3 show high colloid chitin degradation ability than
those of EiChit1 and EhChit1.
Fig. 5.4: Colloidal chitin cleavage patterns of EiChit1, EiChit2, EiChit3 [A] and EhChi1 [B].
Product absorbance intensity was quantified in arbitrary unit (A.U) and plotted in Y axis. The
standard Chitobiose peak is indicated with black arrow. Difference of Y axis scale should be
noted.
97
5.3.4 Inhibitor assay using Methylxanthine compounds
Allosamidine mimics the oxazolinium ring and interact with the conserved aromatic
residues of catalytic cleft. Mostly all family 18 chitinase get inhibited by allosamidine,
as they share the common sequence and TIM barrel structure. Methylxanthine
derivatives were used as drug for treating asthma and inhibiting phosphodiesterase.
Pentoxifylline, Caffeine and Theophylline are such derivatives, with different no. of
attached methyl groups to a xanthine ring (Fig. 5.5)
Fig. 5.5: Chemical structures of Pentoxifylline (A), Caffeine (B) and Theophylline (C)
These three compounds were analyzed in this study, to detect their binding property
and inhibitory effect on Ei chitinases. All three compounds were dissolved in water to
prepare 10 mM stock and used 50-150 µM range for each of the chitinases. 4-
methylumbelliferyl-tri-N-acetylchitotrioside was used as substrate with 200 mM
sodium phosphate buffer (pH-7.2). The product formation was quantified and used for
further calculation. Single substrate-single inhibitor option of Enzyme kinetic module
of Sigma Plot software was used to investigate the inhibition mode. Lineweaver-burk
plot was used to calculate the Ki value (Table 5.2). Pentoxifylline was observed to
show maximum inhibitory effect in general, while caffeine was found to show
minimum Ki values.
The mode of inhibition was determined by plotting the S and V values in competitive,
non competitive and un-competitive inhibition mode available in Sigma plot enzyme
kinetics module (version 9) (Table 5.3).
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Table 5.2: Ki values (µM) of different inhibitors against Ei Chitinases
The best fit inhibition model was selected by comparing the r2 values of different plots,
the accepted plots are presented in appendix C. Interestingly Pentoxifylline, Caffeine
and Theophylline behave in different way for these enzymes, though the three
dimensional structure of these three enzymes are exactly same.
5.4 Discussion
From the computational study it was predicted that all four Entamoeba chitinase
belongs to family 18 with same conserved residues within the catalytic cleft. To
ascertain the predictions, characterization of enzymatic properties of all four chitinases
was done by some well established assays.
Artificial substrate was used to identify the β (1-4) bond cleavage or hydrolytic
efficiency of these proteins. Full length EiChit1 and EhChit1 were found to be less
efficient than EiChit2 and EiChit3, by having a low specific activity and high Km. As
well as they require more incubation period in comparison to EiChit2 and EiChit3.
Binding of small chitotrioside substrate was facilitated by both EiChit2 and EiChit3,
probably due to the absence of the ChBD. The lower efficiency and lower turnover rate
of both EiChit1 and EhChit1 may be explained by the presence of extra domains
(ChBD and the heapta petide repeats).
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All the recombinant chitinases show optimum activity around the pH 7-7.2, for the
artificial substrate with resemblance to the physiological condition in vitro.
Optimum temperature range was determined between 5˚C to 45˚C. While for both
EiChit2 and EiChit3, the optimum temperature was found to be 25˚C, the EiChit1
showed its optimum activity at 37˚C and a reasonable amount of activity from 25°C-
40°C. The maximum activity of EiChit2 and EiChit3 around 25˚C can be explained by
the ambient temperature needed for E. invadens growth in vitro as well as within its
poikilothermal host. So, these enzymes probably get activated within the host body
during excystation. Interestingly, the different behaviour of EiChit1 might be explained
by its localization within the cyst and its two way function. As EiChit1 was detected in
cyst wall, it probably gets exposed to a wide range of temperature. Thus the stability of
EiChit1 within a long range of temperature is needed for its required enzymatic activity
during excystation. EhChit1 shows maximum activity at 37˚C, which resembles its host
temperature.
All recombinant chitinases were assessed to identify their insoluble substrate cleavage
ability, by using amorphous colloidal chitin dust as substrate. All chitinases
predominantly produce chitobiose as exochitinase. Among them, EiChit2 and EiChit3
demonstrate more processivity than both EiChit1 and EhChit1. So, it can be presumed
that, both recombinant EiChit2 and EiChit3 were better enzymes, and cleave their
natural substrate with an ease. The EhChit1 shows the lowest activity against colloidal
chitin.
EiChit1 showed low substrate binding affinity towards the soluble substrate, along with
very slow processivity, which describe it as a poor hydrolytic enzyme, which repeated
with colloidal chitin proving its slow and poor processivity again. Interestingly, the
poor performance of EiChit1 against insoluble substrate may be important, because
during encystation EiChit1 gets transported to the cyst wall, and reside there with chitin
fibrils. In that situation, its low hydrolytic activity against chitin seems to play an
important role without disturbing the cyst wall integrity.
During the substrate binding assay all three Ei chitinases demonstrate enough binding
ability irrespective of the physical difference of three substrates. The chitin binding
efficiency of EiChit2 was comparatively less than that of other two chitinase, as it is
evident from the SDS-PAGE, (Fig 5.3) that though equal amount of chitinases were
100
incubated with equal amount of insoluble chitin, in case of EiChit2 the amount of
bound and unbound enzyme is almost equal, where as in case of EiChit1 and EiChit3
there were no detectable protein in the unbound fractions. The occurrence of binding
ability could be due to the exposed aromatic residues of surface groove of catalytic
domain only, as reported earlier in other prokaryotic chitinases (Suzuki et al., 1999).
From the densitometry scan of band intensity, it was observed that, chitinases binds
most efficiently with chitin beads, which is comparable with colloidal chitin and far
better than chitin dusts. The enzyme binding ability probably depends on the three
dimensional aspects of the substrates and exposed surface also, as the spherical beads
with maximum exposed surface increased the bound protein. Colloidal chitin contains
rough and fibrilar surface which enhance the enzyme binding ability, while the
crystalline chitin dust have lowest amount of available surface and thus less amount of
protein gets bound to it.
It was observed that maximum binding for EiChit2 and EiChit3 occurs at pH 5, while
for EiChit1 it was pH 4. This indicates the involvement of hydrophobic interaction
between the catalytic domain of enzymes and the substrate (Rao et al., 2005).
Three derivatives were observed to inhibit the Ei chitinases, though the inhibition was
found to be around micro molar level as reported previously (Rao et al., 2005).
Pentoxifylline shows lowest Ki values against all three enzymes, which made it the
most potential inhibitor among these three. These derivatives were probably bound
with the aromatic residues of the catalytic domain and mimic the oxazolinium
intermediate.
All three derivatives inhibited each enzyme in more or less similar ways such as either
competitively or non-competitively. Pentoxifylline, Caffeine and Theophylline
inhibited EiChit1 competitively, in spite of their structural differences, which may
explain by the involvement of only the methylxanthine ring. The side groups attached
to the methylxanthine ring probably play no role in the enzyme binding. Among the
three derivatives, Caffeine inhibits two of them competitively, though the Ki values
were high. EiChit2 and EiChit3 were inhibited either non-competitively or
uncompetitively and thus separate themselves from EiChit1. Probably the substrate
binding mode of EiChit1 is different from EiChit2 and EiChit3 in spite of their
101
exceptional similarity in primary sequence and three dimensional structures, and
reflected in the enzymatic kinetics and inhibitor assays of EiChit2 and EiChit3.
From the phylogenetic studies it was concluded that, EiChit3 and EiChit2 originated
before EiChit1, and have more similarity with each other, than with EiChit1. In the
light of enzymatic profile it can be concluded that both EiChit2 and EiChit3 originate
to serve different purpose than EiChit1. Native EiChit1 may have a lower ranking as a
hydrolytic enzyme and primarily work as a polymer connector. The main rationale
behind the evolution of EiChit2 and EiChit3 may explained by their hydrolytic
efficiency rather than their substrate binding ability.
Entamoeba histolytica contain single chitinase, which probably play the both roles as
connector and hydrolytic enzyme in vivo.
5.5 Conclusions
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Chapter 6
6.2 Introduction
On the same note the presence of Chitin binding domain (ChBDs) and putative ChBDs
have been suggested and observed in many chitinases, though detailed molecular and
biochemical analyses of these ChBDs have not yet been reported. Conserved 6-8
cysteine residues and few aromatic residues were found to be the signature sequence
of any ChBD. The penultimate residue, a tryptophan, is supposedly required for
105
interaction with the substrate, and at least two of these conserved residues are required
for binding activity and are predicted to participate in binding to glucosyl units through
hydrophobic or hydrogen bonding (Svensson et al., 1989).
Most of the single or multiple C or N terminal chitin binding domains were further
classified in three domain types, such as ChtBD1 (SMART accession number:
SM00270), ChtBD2 (SMART accession number: SM00494) and ChtBD3 (SMART
accession number: SM00495) depending on their location, structure and amino acid
residues.
Till now 559 Chitin-binding domain type 1(ChtBD1) were identified among 402
proteins and contains approx 43 amino acid residues. This domain resides at the N-
terminus of the mature protein and may occur in one or more copies. A number of
plant and fungal proteins that bind N-acetylglucosamine (e.g. solanaceous lectins of
tomato and potato, plant endochitinases) contain this domain.
106
domain of endoglucanase Z. The domain has a core structure consisting of a 3-
stranded meander beta-sheet containing six aromatic groups.
Entamoeba hsitolytica and Entamoeba invadens were found to contain several chitin
lectins and chitinases containing chitin binding domains having conserved cysteines
which probably form disulphide bridges (Frisardi et al., 2000). Entamoeba chitin
binding lectins, Jacob contains several chitin binding domains with 6 conserved
cysteines residues, while the N terminal chitin binding domain of Entamoeba
chitinases and Jessie were found to have 8 cysteine (Van Dellen et al., 2002) (Fig.
6.1). Few of those putative lectins show several post translational modifications and
proteolytic cleavage sites probably for producing small fragments which required for
substrate binding (Van Dellen et al., 2006). ChBD of Entamoeba chitinases and other
chitin lectins differ in sequence and their activity may be different.
The ChBD of chitinases probably facilitates the accumulation of the enzymes, while
the lectins ChBDs may assist in the formation and maintenance of intricate cyst wall
structure [Van dellen et al, 2006; Chatterjee et al, 2009 (in press)]. As the catalytic
107
domain of all family 18 chitinases were found to be evolutionary conserved, it may not
act as a potential drug or antibody target in human system. Exclusive domain or ChBD
of Eh and Ei may be used as potential target due to its distinctive sequence. EhChBD
show very low structural similarity with EiChBD except of conserved 8 cysteine
residues and few aromatic residues.
6.3 Results
Protein sequences of EiChit1ChBD, other lectin ChBD and other reported ChBDs of
different origin were aligned using Clustal W programme, to identify the probable
substrate binding aromatic residues. Characterization of this ~60 amino acid fragment
could be done by mutagenesis studies of aromatic residues to understand their role.
But the amino acid sequence of EiChit1 ChBD was found to be unique, without
having any similarity with any well studied ChBD, hinder the mutagenesis and
homology studies. So replacement and removal of ChBD is used here to study the role
of EiChit1ChBD in its enzymatic activity.
EiChit1 contains one N terminal 60 amino acid long ChBD, while seven Jacob and
three Jessie lectins contain multiple ChBD and other unidentified domain. Among the
lectins Jacob1 (11-14%) and Jessie3b (18-21%) found to be present in the cyst wall in
comparatively large amount, consisting about 70% of total cyst wall protein along
with chitinase1 (Van Dellen et al., 2006).
EiChit1CAT
108
ChBD of these two lectins (JacobChBD and JessieChBD) were selected due to their high
abundance and probable high substrate binding capacity. Complete removal of ChBD
was done to creating a truncated EiChit1 (EiChit1CAT) (Fig. 6.2).
To understand the expected morphological and other changes, which may occur due to
the modification, primary sequence analyzing tools from www.expasy.org were used
to gather few preliminary information regarding the truncated and fusion proteins.
Theoretical Mol. wt and pI of these three proteins were calculated and tabulated in
Table 6.1.
Table 6.1: Theoretical Mol. wt. and pI of truncated and fusion proteins.
Nucleotide and protein sequences of EiJacob1 and EiJessie3b were obtained from
GenBank.
Primers were designed accordingly to amplify only 180 bp (JacobChBD) and 210 bp
(JessieChBD) long ChBDs of Jacob1 and Jessie3b respectively. Primers were designed for
EiChit1R-CAT (catalytic domain of EiChit1with hepta peptide repeat) and EiChit1CAT
(catalytic domain of EiChit1) also. Forward and reverse primers were designed
incorporating BamHI and BglII sites respectively for JacobChBD and JessieChBD. BglII
and SalI were used in EiChit1R-CAT forward and reverse primers respectively. Primers
for EiChit1CAT fragment were designed by incorporating BamHI and XhoI sites
respectively. Primer sequences were tabulated in materials and methods (Table 3.3).
PCR amplification was done using High Fidelity Expand Taq polymerase (Roche),
and PCR amplified products was verified in 0.8% agarose gel (Fig. 6.3).
109
Fig. 6.3: Amplicons of JacobChBD (180bp), Jessie ChBD (210 bp), EiChit1R-CAT (1296 bp) and
EiChit1CAT (1098 bp). M represents 1kb DNA molecular marker. The amplified products were
indicated by black arrows.
Amplified fragments of JacobChBD, JessieChBD and EiChit1R-CAT were purified from the
gel by gel elution kit (Qiagen, Germany) and cloned primarily in pTZ57R/T vector
(Fermentas) using the T/A overhangs. Truncated fragment EiChit1CAT was cloned in
pGEMT vector (Promega, USA) using the same basic principal. The ligated mixtures
were used to transform E. coli, Dh5α cells for clone selection. Recombinant clones
were selected preliminary by gel retardation study.
110
EiChit1R-CAT [JacChBD (forward), EiChi1R-CAT (reverse) and JessieChBD (forward),
EiChi1R-CAT (reverse) primers] were used to eliminate the incorporation of
homoduplex fragment. Amplified fragments of JacobChBD-EiChit1R-CAT (1476 bp) and
Jessie ChBD-EiChit1R-CAT (1506 bp) were analyzed in 0.8% agarose gel (Fig. 6.4).
Fig. 6.4: Amplified fragments of JacobChBD-EiChit1R-CAT (1476 bp) and Jessie ChBD- EiChit1R-
CAT (1506 bp). M represents 100 bp and 1kb DNA ladders. The amplified products were
indicated with black arrows.
The amplified products were directly ligated to pTZ57R/T for further cloning.
pTZ57R/T clones of JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT were
confirmed with restriction enzyme digestion (Fig. 6.5)
Fig. 6.5: Panel A-Restriction enzyme digested pTZ57R/T clones of JacobChBD-EiChit1R-CAT and
Jessie ChBD- EiChit1R-CAT. Lane 1- Undigested pTZ57R/T -JacobChBD-EiChit1R-CAT clone, Lane
2- Digested pTZ57R/T -JacobChBD-EiChit1R-CAT clone, Lane 3- Undigested pTZ57R/T -
JessieChBD-EiChit1R-CAT clone, Lane 4- pTZ57R/T -JessieChBD-EiChit1R-CAT clone, Lane M- 1kb
DNA ladder. Panel B- Restriction enzyme digested pGEMT-T clone of EiChit1CAT. Lane 1-
Digested pGEMT-EiChit1CAT clone, Lane M-1 kb plus DNA ladder. The inserts are indicated
with black arrows.
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6.3.4 Cloning, Expression and purification of EiChit1CAT, JacobChBD-EiChit1R-CAT
and JessieChBD- EiChit1R-CAT fragments in pQE30 vector
Cloned fusion fragments and truncated fragments were cloned into bacterial
expression vector pQE30 vector for expression purpose. Clone was confirmed by
BamHI and HindIII restriction enzyme digestion. Expression profiles were checked in
E. coli M15 strain after induction with 1 mM IPTG for 5 hr as post induction
incubation period at 37˚C (Fig. 6.6). Comparative analysis was done between un-
induced and induced bacterial culture. Total bacterial protein was boiled with 1X
sample buffer and analyzed in 12% SDS-PAGE with protein molecular wt. marker.
Over expressed protein band of expected mol. wt was observed in the induced lane
only. After separation of membrane bound and soluble protein fraction by sonication
and centrifugation, all over-expressed proteins were mostly found in membrane bound
fragments.
Further confirmation was done by western blot using monoclonal Anti His antibody,
protein A-HRP and DAB detection system (Fig. 6.7). To obtain maximum soluble
protein, optimization of protein expression was done by lowering IPTG concentration
and post induction incubation temperature. Different concentrations of IPTG (0.5-0.02
mM) along with increased incubation period were used for standardization. It was
observed that 0.2 mM IPTG and 16 hr incubation at 15˚C was able to produce
maximum protein in soluble fraction.
112
Fig. 6.7: Western blots of EiChit1CAT, JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT
protein. Monoclonal Anti His Antibody used to detect the bands which are indicated with
black arrows. Approximate molecular weights are also given next to arrow.
Protein purification was done by using Ni-NTA agarose beads and elution was done
with 250 mM imidazole (Fig.6.8). The eluted fractions were analyzed in 12% SDS-
PAGE. Single step purification was found to be enough to purify the protein of interest
to ~80% homogeneity. Purified proteins were further concentrated using 30 kDa cut
off centricon to eliminate the contaminating proteins of low mol. wt. Protein
concentration were determined using Bradford reagent. Purified proteins were used for
further enzymatic studies.
Fig. 6.8: Purified fractions of fusion and truncated proteins. M represents protein molecular
ladder. The purified bands were indicated by black arrows.
113
7.2) and incubated with the purified protein for 15-60 min. The reaction was stopped
by 200 mM sodium carbonate solution and the fluorescence product was measured and
quantified as mentioned before.
It was observed that EiChit1CAT is more active than its parent molecule and JacobChBD-
EiChit1R-CAT need more incubation period than EiChit1, while JessieChBD-EiChit1R-CAT
shows no detectable activity at all, even after 60 min incubation. Kinetics values for
these three fusion proteins were calculated by the Michaelis-Menten equation, using
the enzyme kinetics module of Sigma plot software, and tabulated in table 6.2. Both
EiChit1CAT and JacobChBD-EiChit1R-CAT demonstrate maximum activity against the
soluble substrate at 37˚C and pH 7.2, as their parent molecule.
Both of these fusion proteins show very low/no activity against the soluble substrate,
which may be due to their modified three dimensional structures. To detect the
insoluble substrate cleavage efficiency colloidal chitin and chitin dust was used and
incubated with these fusion proteins. During the enzymatic studies of recombinant
chitinases, colloidal chitin was observed to act as a more accessible substrate due to
the presence of more open ends. Crystalline structure of chitin dust was found to be
more compact in nature and need long incubation period.
The elution profiles were plotted using multi-curve option of Origin 8 software and
plotting the absorbance values at Y axis. Both JacobChBD-EiChit1R-CAT and JessieChBD-
EiChit1R-CAT demonstrate cleavage ability against colloidal chitin. JacobChBD-EiChit1R-
CAT was found to produce comparatively more oligomer product than JessieChBD-
EiChit1R-CAT from colloidal chitin within same incubation period.
A time dependent study was done by studying the supernatant obtained from reaction
mixtures of different incubation period (2-24 hr). Product analysis was done as earlier
(Fig. 6.10).
115
Fig. 6.10: Time dependent profiles of chitin dust degradation by JacobChBD-EiChit1R-CAT [A]
and JessieChBD-EiChit1R-CAT [B]. Product absorbance intensity was quantified in arbitrary unit
(A.U) and plotted in Y axis. Chitobiose standard was marked by black arrow. Difference in
scales should be noted.
Binding assay was performed as described before and the bound and unbound
fractions were analysed in 12% SDS-PAGE, followed by staining and destaining (Fig
6.11A). Densitometry scan of SDS-PAGE was done to calculate the binding efficiency
and values are plotted on a graph (Fig 6.11 B). It was observed that, both proteins bind
preferentially to colloidal chitin, while the least binding occur with chitin dust.
Maximum substrate binding occur at different pH range, which coincide with their pI
values.
To recognize the maximum substrate binding affinity of the ChBD, both fusion
proteins and the native one were incubated with fixed amount (100 µl of suspension)
of chitin beads for 2 hr with constant agitation. Free and bound protein was separated
by centrifugation, and the free protein content of the supernatant was measured. Bound
protein fraction was calculated by subtracting the free protein amount from initial
116
amount. The values were plotted using Langmuir equation, using single site ligand
binding mode of Sigma plot software. Dissociation constant was calculated to identify
the best ChBD. It was observed that JessieChBD-EiChit1R-CAT bound most efficiently to
chitin beads. EiChit1 was found to be a little less competent than JessieChBD-EiChit1R-
CAT, but perform far better than JacobChBD-EiChit1R-CAT at substrate binding.
Fig. 6.11: SDS-PAGE analysis of substrate binding ability of fusion protein JacobChBD-
EiChit1R-CAT and JessieChBD-EiChit1R-CAT [A]. Graphical representation of percentage of bound
protein from densitometry scan values [B].
6.4 Discussion
All three Ei Chitinases show chitin binding ability in presence or in absence of specific
chitin binding domain, as the catalytic domain also bind to chitinious material to some
extent. But from the point of identifying new drug target, the catalytic domain of was
not found to be of good choice because of their conserved sequences throughout the
evolution. The ChBD of Ei chitinase 1 was found to be an exclusive domain, without
having any similarity with human ChBD. EiChit1ChBD doesn’t have any striking
similarity with EhChit1ChBD though the key cysteine and aromatic residues were found
to be placed strategically.
As the aromatic residues were believed to play an important role in substrate binding,
it was crucial to identify the important residues within EiChit1ChBD. To recognize these
key aromatic residues, wild EiChit1ChBD was replaced with two other lectins ChBDs
having six (JacobChBD) and eight cysteine (JessieChBD) residues. Two fusion proteins
JacobChBD-EiChit1R-CAT and JessieChBD-EiChit1R-CAT were created by restriction
117
enzyme digestion and PCR amplification. Expression of fusion proteins were done in
bacterial system.
Previously it was observed that EiChit1 show slow hydrolytic activity as well as low
substrate affinity towards soluble substrate which was probably due to the presence of
extra domain. So to reveal the role of this extra domain, a truncated EiChit1 was
expressed. Both fusion proteins are found to have lowered pI in comparison their
parent protein EiChit1, while truncated portion retain the same pI. So it can be
presumed that the removal of 60 N-terminal amino acids does not have any significant
effect in the total charge of the protein, while the addition of extra 60 amino acids
change the total charge towards the positive values probably making the fusion
proteins little more basic.
All fusion and truncated proteins were tested against soluble substrate and kinetic
profiles were calculated. Truncated EiChit1CAT shows high hydrolytic activity and
high substrate affinity as expected. Absence of ChBD was found to be helpful for
proper substrate binding and enzymatic processing in case of soluble substrate. Native
EiChit1 show lower activity against insoluble substrates and found on the cyst wall of
encysting and mature cyst, probably helping the binding of chitin fibrils, which
evidently doesn’t require high hydrolytic activity against the fibrils. It is tempting to
conclude that, EiChit1 was evolved not only for cleaving the chitin fibrils at large but
also to assemble them in a tightly knitted structure.
As the soluble substrate doesn’t resemble the natural condition, both the fusion
proteins were tested against colloidal chitin and chitin dust. Long incubation period
was needed to produce sufficient product, which belongs to chitobiose group. Both
fusion proteins were efficiently cleave loosely integrated colloidal chitin and compact
chitin dust. JacobChBD-EiChit1R-CAT cleaves colloidal chitin more in comparison with
JessieChBD-EiChit1R-CAT. But in the case of chitin dust, JessieChBD-EiChit1R-CAT were
118
found to be more competent than JacobChBD-EiChit1R-CAT by releasing more dimers
within 2 hr. After a long incubation period JessieChBD-EiChit1R-CAT release more
chitobiose than JacobChBD-EiChit1R-CAT. In both cases, preliminary profiles were
shown to contain high molecular weight product, which later masked by accumulating
chitobiose.
Substrate binding affinity of these two fusion protein increased at their pI, indicating
hydrophobic interaction as the interacting force. Substrate binding ability of these two
fusion proteins were compared with native EiChit1. JacobChBD-EiChit1R-CAT and
JessieChBD-EiChit1R-CAT were found to exhibit lowest and highest binding affinity
respectively towards chitin beads, while EiChit1 show intermediate affinity.
The alignment of all three ChBD (Fig. 6.12) reveal some interesting points, such as
EiChit1ChBD contain more number of aromatic residues than JacobChBD which
strengthen the relationship between aromatic residue number and substrate binding
affinity (Kikkawa et al., 2008). Few conserved aromatic residues were identified
between EiChitChBD and JessieChBD. After aligning the EhChit1ChBD sequence, those
aromatic residues were again found to be conserved. Alignment with Human ChBD
shows no significant sequence similarity with EhChit1ChBD, which increase its
potential as a drug target.
It can be observed that three ChBD sequences bear few conserved aromatic residues,
though they were more conserved between EiChit1ChBD and JessieChBD and placed
strategically. Among the residues, Phe8, Try18, Try49 and Try51 probably play the
most important role in substrate binding ability.
Fig. 6.12: Entamoeba chitinase and lectin ChBD aligned and compared with human ChBD.
Conserved cysteine were coloured in red and aromatic residues coloured in yellow. Aromatic
residues responsible for substrate binding are indicated with black arrows.
119
6.5 Conclusions
• Fusion protein containing different chitin binding domain were designed and
expressed in bacterial system.
• Truncated EiChit1CAT was created by removing the ChBD and expressed in
bacterial system.
• Truncated EiChit1CAT exhibit more hydrolytic ability indicating that the ChBD
is not required to hydrolyze small substrates.
• Both fusion proteins were able to hydrolyze insoluble substrates though they
behave differently with soluble substrate.
• Number of Cysteine and aromatic residues and their placement within the
ChBD probably play some important role in maintaining the structural integrity
and substrate binding ability respectively.
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Chapter 7
7.2 Introduction
123
with the visible morphological changes, many genes were observed to get regulated at
transcriptional level during this process (Sanchez et al., 1994). Several encystation
specific genes such as Jacob (Frisardi et al., 2000), chitinase (Villagomez-Castro et al.,
1992) and chitin synthase (Das and Gillin, 1991) were found to be up regulated in Ei.
Only 15% of Ei genes were predicted to be regulated developmentally during this
transformation. All these genes have their respective homologues in Eh, and they are
also found to up-regulated in isolated clinical strains of xenic culture of E. histolytica,
grown in complex diphasic media (such as Robinson’s medium with an agar slant)
(Ehrenkaufer et al., 2007).
In this report, an effort was made to characterize the transcriptional and localization
profile of all three Ei chitinases.
7.3 Results
Encystation was standardized using log phase E. invadens cell. Cells were washed
thrice and resuspended in 47% LG (low glucose) medium (Sanchez et al., 1994). After
36-48 hr clumping of Ei cells occur with appearance of spherical cyst (Fig. 7.1 C and
124
D). Within 60-72 hr of encystation 60-70% encystation was observed, though the
encystation was not found to be synchronic.
Fig.7.1: Confocal micrograph of E. invadens trophozoites and cyst. Fluorescence (A) and
phase contrast (B) image of Ei trophozoites with DAPI stained single nucleus. The bar in A
and B represents 5 µm. Fluorescence (C) and phase contrast (D) image of Ei cyst with DAPI
stained four nuclei. Nuclei are indicated with white arrows. The bar in C and D represents
2 µm.
Fig.7.2: RT-PCR analysis of Ei Chitinase 1, Chitinase 2 and Chitinase 3 at different time point
of encystation. Lane DNA in each panel represent the chitinase amplicons amplified from
genomic DNA (+ve control). Hours are representing the encystation time period. Ei Chitinase
2 PCR product was loaded in double amount with respect to other chitinases (indicated by 2x)
to get a cooperative image. Actin amplification was used as internal control.
125
Total RNA was isolated using TRI Reagent and was used for cDNA synthesis using
oligo (dT) primers by RETRO script first strand synthesis kit (Ambion). PCR was
carried out with cDNA using chitinase gene specific primers, designed to amplify only
~400 bp region. Actin was used as an internal control and all the amplicons were
analyzed in 1.5% agarose gel (Fig.7.2).
All three Ei chitinase proteins bear a very high sequence similarity within their
catalytic domain. So, it was presumed that a single antibody raised against the
catalytic domain may identify three chitinases in vivo. Polyclonal antibody in rabbit
was raised by following the method described in Method and Material. Briefly,
EiChit2 (only the catalytic domain) was purified using Ni-NTA bead and analyzed in
12% SDS-PAGE. Electro-elution of purified EiChit2 from the sliced gel piece was
done for further purification. Lyophilized protein was used for subsequent
intramuscular injections. Finally 12 ml blood was collected by heart bleed and used
for IgG purification. Western blot and ELISA were done against recombinant EiChit2
to detect antibody specificity and antibody titre. Anti EiChit2 antibody was observed
to identify all three recombinant Ei chitinases.
126
secondary antibody. Detection of bands was done with DAB system. Two distinct
bands of approximately 35 and 50 kDa were identified after 60 hr of incubation.
Fig.7.3: Western blot of total protein isolated from mature Ei cysts of 60-72 hr. Anti EiChit2
antibody was used as primary antibody. Panel A- Lane1-3 represents silver stained SDS-
PAGE of 0 hr, 60 hr and 72 hr respectively. Lane M represents the protein mol wt markers.
Panel B - Lane 1-3 represents developed blot of 0 hr, 60 hr, and 72 hr respectively. The
developed bands were indicated with black arrows. Two distinct bands of 35 and 50 kDa were
observed.
For microscopic study encysting Ei cells of different time period (0- 72 hr) was chilled
and fixed with 2% PFA. Permeabilization was done by 0.01% Triton X-100.
Permeabilized and non-permeabilized cells were incubated with anti chitinase
antibodies for 1 hr (1:200 dilution), followed by the washing with chilled 1X PBS for
three times. Secondary staining was done with TRITC conjugated anti rabbit IgG
(1:500 dilution). Stained cells were visualized with Olympus FluoView FV1000
confocal microscope with TRITC channel. All the images were taken in Z-stacking
mode and the middle slice was used for representation. Image analysis and
representation was done using FluoView software (Olympus) (Fig.7.4 and Fig.7.5).
Anti EiChit2 antibody also detect chitinase in maturing cyst wall, which might be due
to the presence of Ei Chitinase 1 as this is also detected by anti EiChit1 antibody and
Ei Chitinase 1 only has the chitin binding domain.
127
Fig.7.4: Fluorescence (bottom panel) and phase contrast (upper panel) image of Ei Chitinase 1
localization in encysting E. invadens at different time point. Encysting cells were fixed,
permeabilized and incubated with Ei Chitinase 1 primary antibody and TRITC conjugated anti
rabbit IgG secondary antibody. Panel A-F represent cells of 0 hr, 24 hr, 36 hr, 48 hr, 60 hr and
72 hr incubation. The bar represents 5µm.
Anti EiChit2 antibody detects all three chitinases to some extent, and identifies Ei
Chitinase 1 in cyst wall (Fig. 7.5). EiCht2 and EiChit3 were not found in cyst wall
analyzed by Mass spectrometry (Van Dellen et al., 2006). In mature cyst small
vesicles within the cyst wall were detected by anti EiChit2 antibody, which was not
localized by anti Ei Chitinase 1 antibody that indicates the presence of only Ei
Chitinase 2 and Ei Chitinase 3 in those vesicles of mature cysts. Much better signal
obtained by anti EiChit2 antibody over anti EiChit1 antibody is probably due to high
affinity and better quality.
128
Fig.7.5: Fluorescence (bottom panel) and phase contrast (upper panel) image of Ei chitinase
localization in encysting E. invadens at different time point. Encysting cells were fixed,
permeabilized and incubated with EiChit2 primary antibody and TRITC conjugated anti rabbit
IgG secondary antibody. Panel A-H represent cells of 0 hr, 12 hr, 18 hr, 24 hr, 36 hr, 48 hr, 60
hr and 72 hr incubation. The bar represents 5µm.
From the protein sequence alignment of all Entamoeba chitinases it was observed that
catalytic domain of EhChit1 has high resemblance with EiChit2. So it was assumed
that the polyclonal antibody raised against EiChit2 catalytic domain may detect the Eh
counterpart also. Western blot was done to identify the cross recognition level, and
anti EiChit2 antibody was found to recognize recombinant EhChit1 (Fig.7.6). Eh cyst
in clinical stool samples was collected from symptomic patients of surrounding
129
hospitals and clinics. Collected samples were diluted with 1X PBS and filtered
through steel mesh to discard the debris.
Fig.7.6: Western blot analysis of recombinant EhChit1 using anti EiChit2 antibody. EiChit2
and EhChit1 bands are detected and indicated by the black arrow. M represents the pre-stained
protein marker and mol wt of only visible bands is represented here.
Filtrate fractions was collected and washed with 1x PBS for twice. After confirming
the presence of mature Eh cyst by phase contrast microscopy, the samples was
incubated with anti EiChit2 antibody for 1 hr followed by 1hr incubation with TRITC
conjugated anti rabbit IgG. Counter staining of chitin wall was done by incubating the
samples for 30 min at room temperature with 10 µl of 100 mM Calcoflour White
(CFW) stain. Stained samples were visualized with Olympus FluoView FV1000
confocal microscopy using TRITC and DAPI channel for TRITC and CFW stain
respectively (Fig.7.7). Images were taken in Z-stacking mode and the middle slide was
represented in TRITC panel.
Anti EiChit2 antibody was able to recognize the Eh Chitinase 1 in mature Eh cyst
wall, and can be used for clinical detection of Eh cyst.
Fig.7.5: Fluorescence image of Eh cyst stained with Calcoflour White (A) and Anti EiChit2
antibody (B). The bar represents 2 µm.
130
7.4 Discussion
Human gastro-intestinal tract parasite such as Giardia and Entamoeba, live a very
simple yet opportunistic life within the host. E. histolytica enter the host body in a
chitin walled cyst form, through contaminated food or water and gives rise to four
trophozoites. The excystation occur within the alkaline environment of small intestine.
Trophozoites invade the mucous and sub mucous layer of the small intestine and cause
the well known symptoms such as irritable bowel syndrome, and bloody mucous.
Though very few cyst bearers ever become cyst passers, at a certain time point the
existing trophozoites in large intestine enter the encystation cycle triggered by some
unknown stimulants. For Giardia most of the physical conditions such as cholesterol
starvation, responsible for such changes were discovered (Lujan et al., 1996) while for
E. histolytica the triggers are unknown (Eichinger, 2001).
E. invadens, a reptilian parasite was used widely as a model for encystation study, due
to its capacity to encyst in vitro. Low nutrition, high concentration of parasite and high
osmotic pressure were found to trigger the encystation in vitro for E. invadens
(Sanchez et al., 1994) however the same conditions were not found enough to induce
the changes in E. histolytica. Very complex procedure of E. histolytica encystation
was reported several times, despite the fact that none of them were able to encyst
axenic culture (Ehrenkaufer et al., 2007).
After the initiation, the exact order of cyst wall formation or the appearance of
different encystation specific protein are not clear, though most important players of
this change are being characterized. Jacob, a multi-domain chitin binding lectin was
discovered to appear first (Frisardi et al., 2000), followed by a string of other chitin
binding proteins such as chitinase and Jessie. Chitin and chitosan were found as the
main ingredients of the polysaccharide constituent (Das et al., 2006). A complex
meshwork of carbohydrate and protein are formed to cover the cyst structure. As a
part of the protein complex, multiple chitin binding lectins such as Jacob and Jessie
with different domain structure along with the chitinase play an important role to
complete the meshwork (Chatterjee et al., 2009) (in press).
131
chitinase with an extra chitin binding domain detected in the cyst wall, while the trace
of other two chitinases were not detected either in cyst wall or in m RNA pool of
previous studies (Van Dellen et al., 2006)
In this study, encysting cells of different time period shows the presence of all three
chitinases from 18 hr onwards which continued up to 48 hr. All three chitinases get
transcribed in more or less similar fashion, though the transcription of Ei Chitinase 2
stopped first at 36 hr and expressed in lesser quantity than that of other two chitinases
at least in transcription level.
Polyclonal anti EiChit2 antibody was raised and found to detect all three recombinant
Ei chitinases during immunoblotting. Total protein from mature Ei cysts of 60-72 hr
was collected to detect the presence of native chitinases. Two distinct bands of 35 and
50 kDa (approx) were noticed after 60 hr of encystation. Previous studies with native
Ei chitinases reports same range of mol wt. for Ei Chitinase 1 (approx 50 kDa) and Ei
Chitinase 2 and 3 (both around 35 kDa) (Villagomez Castro and Lopez-Romero,
1996). Though the signals were found to be poor, it can be concluded that anti EiChit2
antibody recognize other two native chitinases as well.
Localization of Ei Chitinase 1 specifically was done by the antibody raised against the
repeat region; while anti EiChit2 antibody identify all three chitinases as all three of
them share a similar catalytic domain. During the time based study it was observed
that, Ei Chitinase 1 translated and appear along with other two chitinases. Translated
proteins were accumulated within numerous small vesicles and distributed throughout
the cytoplasm of encysting cells. Within 24 hr of encystation Ei Chitinase 1 was
observed to reach the cyst wall, which indicates that it might play a role in chitin
binding and cyst wall formation. Vesicles containing Ei Chitinase 2 and 3 were also
observed to reach the area adjacent to cyst wall. Large vesicles were detected by this
antibody around the cyst wall, which may contain Ei Chitinase 2 and 3, as they were
not separated in this study. Vesicles containing electron dense material surrounding
the cyst wall were reported previously (Chavez-Munguia et al., 2003), concluded to
contain wall dissolving enzymes. From the present study it can be concluded that
those vesicles contain chitinase which may help to degrade the chitin wall from inside
during excystation. No vesicular localization of Ei Chitinase 1 was not detected in
mature cyst, which indicates that the Ei Chitinase 1 presents only in the cyst wall and
132
play the major role during the encystation to made the tailor made chitin fibrils. Which
was not possible to conclude from this study is whether Ei Chitinase 2 or Ei Chitinase
3 is present in the cyst wall or not. Different chitinase specific antibody might resolve
this question.
Due to the high similarity of protein sequence Anti EiChit2 antibody was able to
identify other two recombinant as well as native Ei chitinases also. As EiChit2 share a
common catalytic domain with EhChit1 also, it was presumed that, the antibody may
identify the recombinant EhChit1 also. From the western blot it was discovered that,
indeed the antibody recognize the Eh chitinase. Eh Chitinase 1 in mature Eh cyst was
also detected by the anti EiChit2 antibody, confirming the presence of Eh Chitinase 1
on the cyst wall. This cross recognition may be used to identify clinical sample.
7.5 Conclusion
133
134
Chapter 8
Discussion
136
Chitin as a structural polysaccharide placed 2nd most abundant polymer on Earth after
cellulose and play an important role in bacterial, fungal, insect and higher vertebrate
life, by creating the their cell wall or exoskeleton. Natural pool of chitin remains same
though it is insoluble in most of the available solution. Chitinase, a member of
glycosyl hydrolase group is responsible for the biodegradation of chitin and found to
occur in most of the chitin bearing organism, as well as in the non-chitinious
organisms also. Chitinases are assigned many roles ranging from food processing to
immuno-modulation depending on the system. Occurrences of multiple chitinases
were observed in many bacterial and fungal species, where they act synergistically to
degrade the polymeric chitin into soluble oligomers. Origin of multiple chitinase and
chitinase like proteins were assumed due to domain shuffling and gene duplication.
Phylogenetic analysis of chitinases depicts their conserved structural aspects
throughout the evolutionary pathway.
Among the human protozoan parasites, E. histolytica is responsible for more than 48
million new infections and more than 100,000 deaths per anum (Walsh, 1986). Current
treatment system of amoebiasis largely depends on a nitro-imidazole drug
“Metronidazole” (Mtz) only. Occurrence of Mtz resistant bacterial and amoebic strains
in last few years initiated the search for new drug targets in Entamoeba, which was
accelerated by the release of E. histolytica genome project (Loftus et al., 2005).
Among other plausible targets, encystation and excystation specific molecules were
found to be of heightened interest, as their inhibition can restrain the infection of new
hosts as well as invasion of host gastro-intestinal tract respectively. E. invadens, a
reptilian parasite resemble the human parasite and used worldwide as a model of
encystation study. Advancement in genomic studies of E. histolytica, disclose the
137
presence of a single chitinase gene, similar to Entamoeba dispar, though the reptilian
counterpart was reported to contain three chitinases (Wang et al., 2003).
Inhibitor assay was done using methylxanthine derivatives as lead inhibitor molecules.
Methylxanthine derivatives were supposed to mimic the oxazolinium intermediate
within the catalytic domain of family 18 chitinases as allosamidine, the most potent
chitinase inhibitor (Rao et al., 2005). It was observed that all three derivatives such as
Pentoxifylline, Caffeine and Theophylline inhibit EiChit1 in competitive way, in spite
of their different side chain structure. It can be assumed then, that the side chains are
not important for enzyme binding or inhibition. Both EiChit2 and EiChit3 were found
to be inhibited in uncompetitive or in-competitive way, exhibiting their different
substrate binding mode. From the different inhibition profile and Ki values, it was
found that pentoxifylline was the most potential lead molecule among the three and
EiChit1 get inhibited maximum by following the family 18 substrate binding mode
more efficiently.
ChBD was discovered in human chitinase without any significant similarity with
Entamoeba ChBD. This amino acid long fragments were assumed to enhance the
substrate degradation by increasing enzyme concentration on polymeric substrate
surface. To identify the role of 8 cysteine rich ChBD of EiChit1, it was replaced by
two other E. invadens lectin ChBD of 60 (JacobChBD) and ~70 (JessieChBD) amino
acids. Truncated EiChit1 (catalytic domain only) was created to detect the role of
EiChit1ChBD on its own enzymatic profile. Fusion proteins were created by restriction
139
enzymatic digestion and PCR amplification. The truncated protein exhibits increased
hydrolytic activity against soluble substrate, explaining the lowered efficiency of
EiChit1, due to the presence of extra domain. Both fusion proteins exhibited somewhat
diminished/no activity against soluble substrate, which may be due to different
substrate binding mode. But hydrolysis of insoluble substrate was not found to be
impeded due to the replacement of ChBD. Both fusion and native proteins exhibit high
substrate binding ability and JessieChBDEiChit1R-CAT shows maximum binding ability,
followed by EiChit1 and JacobChBDEichit1R-CAT. Analysis of ChBD sequence reveals
that, EiChit1ChBD contains maximum aromatic amino acids, which may directly
influence the substrate binding ability (Kikkawa et al., 2008). Multiple alignment of
all three ChBD identifies few strategically placed aromatic residues in both JessieChBD
and EiChit1ChBD, which are assumed to play important role in substrate binding. It was
further observed that, EhChit1ChBD also contains those specific residues, probably
playing the same role. So, it can be concluded that ChBD might be to EiChit1 and
EhChit1 moderate its hydrolytic activity but to increase its substrate binding activity.
The sequence of EiChit1ChBD might be tuned evolutionarily to optimize the substrate
binding, without obstructing the substrate hydrolysis.
140
wall. From the presence of chitinase containing vesicles surrounded by the cyst wall
and the absence of both EiChit2 and EiChit3 in cyst wall protein (Van Dellen et al.,
2006), it can be concluded that the EiChit1 exist on the wall and the EiChit2 and
EiChit3 probably reside within those vesicles and not on the cyst wall, as both of them
lack the ChBD. It can also be assumed that, EiChi1 exist on the cyst wall, while
EiChit2 and EiChit3 stay within the vesicles adjacent to cyst wall waiting for the
excystation specific signals.
Finally we conclude that all three Ei Chitinases were evolved to play different roles
during the encystation and excystation. EiChit1, EiChit2 and EiChit3 simultaneously
get transcribed and translated during encystation. They get transported through
vesicles towards the cyst wall, where EiChit1 reach the cyst wall, while other two
remain within the vesicles. During encystation EiChit1 trim the chitin fibrils and help
in cyst wall formation, while other two more efficient enzymes help during
excystation by cleaving the chitin wall.
Evolutionarily they evolved in different time point, following the order of EiChit3,
EiChit2 and EiChit1. The most currently evolved protein EiChit1 coincide with
EhChit1 in every way. So, it may be assumed that, previously E. invadens used to
have more efficient single chitinase which get multiplied either by gene duplication or
domain shuffling through evolution, parallel to EhChit1.
141
142
Chapter 9
6. Two Fusion proteins were created using lectin ChBD and catalytic domain
of EiChit1 and cloned along with EiChit1CAT followed by bacterial
expression using pQE30 vector.
12. EiChit1 detected very feebly on the mature cyst wall, while anti EiChit2
antibody detects chitinases in cyst wall and small vesicles.
13. Anti EiChit2 antibody was able to identify recombinant and native
EhChit1, and detect mature Eh cyst in clinical samples.
145
9.2 Future Scopes
146
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Appendices
Appendix A
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Appendix B
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Appendix C
Fig. I: Lineweaver Burk Plot of EiChit1 with 1/S along X-axis and 1/V along Y-axis.
These graphs display the inhibition mode of EiChit1 by Pentoxifylline, Caffeine and
Theophylline. Increasing concentrations of inhibitors (50-150 µM) were used with 1-5 µM
range of 4-methylumbelliferyl-tri-N-acetylchitotrioside as substrate. The kinetic data were
plotted in single substrate-single inhibitor mode of Enzyme kinetics module of Sigma plot (9)
software. Every data set was checked for full competitive, full non-competitive and full un-
competitive inhibition mode and the plot with highest r2 value is represented here.
161
Fig. II: Lineweaver Burk Plot of EiChit2 with 1/S along X-axis and 1/V along Y-axis.
These graphs display the inhibition mode of EiChit2 by Pentoxifylline, Caffeine and
Theophylline. Increasing concentrations of inhibitors (50-150 µM) were used with 1-5 µM
range of 4-methylumbelliferyl-tri-N-acetylchitotrioside as substrate. The kinetic data were
plotted in single substrate-single inhibitor mode of Enzyme kinetics module of Sigma plot (9)
software. Every data set was checked for full competitive, full non-competitive and full un-
competitive inhibition mode and the plot with highest r2 value is represented here.
162
Fig. III: Lineweaver Burk Plot of EiChit3 with 1/S along X-axis and 1/V along Y-axis.
These graphs display the inhibition mode of EiChit3 by Pentoxifylline, Caffeine and
Theophylline. Increasing concentrations of inhibitors (50-150 µM) were used with 1-5 µM
range of 4-methylumbelliferyl-tri-N-acetylchitotrioside as substrate. The kinetic data were
plotted in single substrate-single inhibitor mode of Enzyme kinetics module of Sigma plot (9)
software. Every data set was checked for full competitive, full non-competitive and full un-
competitive inhibition mode and the plot with highest r2 value is represented here.
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Curriculum vitae
Tuli Dey
Microbiology and Immunotechnology Laboratory
Department of Biotechnology
Indian Institute of Technology, Kharagpur
India 721302
E mail - tulidey@yahoo.com
Phone no. +91-3222-255226 (Res)
+91-9474143894 (Mob)
Educational Qualifications
• Doctoral studies from 2005 in Molecular Parasitology in the Department of
Biotechnology, Indian Institute of Technology, Kharagpur West Bengal India.
• Masters in Science (Zoology) from Vidyasagar University, West Bengal, India
(2000).
• Bachelor in Science (Zoology, chemistry and physiology) from Raja N.R.L.
Khan Women’s college. West Bengal, India (1998).
Awards
• Awarded Senior Research fellowship from CSIR, India in 2007
• Qualified National Eligibility Test in Life Sciences and awarded Junior
Research fellowship from CSIR, India in 2004.
• Qualified Graduate Aptitude Test in Engineering (GATE) in 2003.
• Qualified Graduate Aptitude Test in Engineering (GATE) in 2002.
Publications
• Dey, T., Basu, R., Ghosh, S.K., Entamoeba invadens: Molecular
characterization of chitinases (under review).
• Pal, T.K., Dey, T., Ghosh, S.K., Pathak, T., First synthesis and antiprotozoal
activities of Divinyl sulfone-modified carbohydrates (communicated)
Conference presentation
• Dey, T., Basu, R., Pathan, M., Ghosh, S. K.
Molecular characterization of Entamoeba invadens Chitinase 2
Organized by 19th Molecular Parasitology Meeting 2008 MBL, Woods Hole,
Boston.
• Dey, T., and Ghosh, S. K.
Cloning and molecular modelling of Entamoeba Chitinases.
Oral presentation: “National Seminar on “Microbes in Pharmaceuticals, Food
& Agriculture” Organized by Department of Microbiology Vidyasagar
University, Midnapore-721102, West Bengal. December 20 – 21, 2006.
• Pal, D., Dey, T., Sameulson, J., Ghosh, S. K.
Molecular Characterization of Entamoeba histotytica Nitroreductase.
Poster presentation: 74th Annual meeting of Society of Biological Chemists
[SBC], Central Drug Research Institute, Lucknow, India. November 7-10,
2005.
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